A 

DeM, 


THE  PROTEINS  OF  THE  WHEAT  KERNEL 


BY 


THOMAS   B.  OSBORNE 


WASHINGTON,  D.  C. : 

Published  by  the  Carnegie  Institution  of  Washington 
1907 


THE  PROTEINS  OF  THE  WHEAT  KERNEL 


BY 


THOMAS   B.  OSBORNE 


WASHINGTON,  D.  C. : 

Published  by  the  Carnegie  Institution  of  Washington 
1907 


Mam 


CARNEGIE  INSTITUTION  OF  WASHINGTON 
PUBLICATION  No.  84  , 


PRESS  OF  JUDD  &  DETWEILER,  INC. 
WASHINGTON,  D.  C. 


CONTENTS. 


Page. 

INTRODUCTION 5 

REVIEW  OF  THE  LITERATURE 6 

EXPERIMENTAL  PART 17 

The  aqueous  extract  of  wheat  flour 18 

The  albumin  of  wheat  flour — leucosin 20 

The  aqueous  extract  of  the  wheat  embryo 22 

Hydrolysis  of  leucosin 30 

The  proteins  of  wheat  flour  soluble  in  sodium-chloride  solution 37 

The  globulin  of  wheat  flour 37 

The  proteins  of  the  wheat  embryo  soluble  in  sodium-chloride  solution 40 

The  globulin  of  the  wheat  embryo 40 

The  proteose  of  wheat  flour 45 

The  proteose  of  the  wheat  embryo 46 

J  The  proportion  of  the  various  protein  substances  of  the  wheat  embryo 48 

Digestion  of   the  phosphorus-containing  protein  preparations  with  pepsin- 
hydrochloric  acid , 49 

Protein  soluble  in  dilute  alcohol — gliadin 53 

Direct  extraction  with  dilute  alcohol 53 

Extraction  with  dilute  alcohol  after  extracting  the  flour  with  10  per  cent 

sodium-chloride  solution 58 

\    Extraction  of  gluten  with  dilute  alcohol 59 

Extraction  of  "  shorts  "  with  dilute  alcohol 60 

Protein  extracted  by  alcohol  from  whole-wheat  flour 62 

The  proportion  of  glutaminic  acid  yielded  by  various  fractions  of  the  alcohol- 
soluble  protein 64 

Hydrolysis  of  gliadin 70 

Protein  insoluble  in  water,  saline  solutions,  and  alcohol — glutenin 82 

Protein  extracted  by  dilute  alkaline  solutions  after  extracting  the  flour 

with  10  per  cent  sodium-chloride  brine  and  then  with  dilute  alcohol. ...  82 
Protein  extracted  by  dilute  alkaline  solutions  from  gluten  after  extracting 

the  latter  with  dilute  alcohol 84 

Protein  extracted  by  dilute  alkaline  solutions  after  complete  extraction 

with  dilute  alcohol  of  gluten  from  whole- wheat  flour 88 

Hydrolysis  of  glutenin 91 

A  The  amount  of  the  various  proteins  contained  in  the  kernel  of  wheat 100 

The  formation  of  gluten 103 

SUMMARY 108 

Gliadin 109 

Glutenin 112 

Leucosin 113 

The  globulin 115 

The  proteose 116 

The  gluten 116 

The  nutritive  value  of  the  wheat  proteins 117 

3 


398097 


THE  PROTEINS  OF  THE  WHEAT  KERNEL. 


INTRODUCTION. 

Of  the  protein  substances  used  as  food  none  is  of  more  importance  than 
those  contained  in  the  seeds  of  wheat.  Although  these  bodies  attracted 
the  attention  of  investigators  more  than  one  hundred  years  ago  and  have 
since  then  been  many  times  the  subject  of  study,  the  published  statements 
respecting  them  are  so  conflicting  and  uncertain  that  it  has  heretofore  been 
impossible  to  know  what  the  truth  regarding  them  actually  is.  With  the 
purpose  of  clearing  up  the  existing  confusion  and  determining  the  real 
value  of  the  evidence  offered,  as  well  as  extending,  as  far  as  possible,  our 
knowledge  of  these  important  substances,  the  writer  some  years  ago  under- 
took an  investigation  of  this  seed  which  has  recently  been  concluded  by 
work  done  under  grants  from  the  Carnegie  Institution  of  Washington.  As 
the  results  of  these  investigations  have  been  published  from  time  to  time  in 
a  number  of  different  papers,  appearing  in  four  different  journals,  it  has 
been  thought  desirable  to  bring  all  this  work  together  in  one  paper.  In  so 
doing  the  details  have  been  reproduced  in  full,  for  the  nature  of  the  evidence 
is  such  that  its  value  largely  consists  in  concordant  results  of  many  experi- 
ments, repeated  under  different  conditions,  since  it  is  not  yet  possible  to 
establish  the  chemical  individuality  of  different  protein  substances  by  demon- 
strating their  possession  of  definite  physical  properties,  as  may  be  done  with 
the  simpler  organic  compounds. 

The  experience  of  the  writer  in  his  endeavors  to  understand  and  repeat 
the  work  of  many  of  his  predecessors  has  made  him  feel  the  importance  of 
these  details  to  future  workers  along  the  same  lines  and  is  his  excuse  for 
giving  with  so  much  minuteness  the  results  of  his  own  work,  which  to  those 
not  familiar  with  the  difficulties  of  the  subject  must  appear  to  a  large  extent 
unnecessary.  In  order  to  make  this  work  available  to  those  who  wish  sim- 
ply to  know  the  results,  a  comprehensive  summary  of  this  paper  is  given  at 
the  end  of  this  publication,  and  the  details  of  the  many  operations  and  experi- 
ments need  be  read  only  by  those  who  wish  to  become  familiar  with  these. 

The  account  of  this  work  is  preceded  by  a  review  of  the  literature  of  the 
subject,  from  which  an  idea  of  the  unsatisfactory  state  of  our  previous 

5 


6  THE;  PROTEINS  OF  THE  WHEAT  KERNEL. 

knowledge  can  be  obtained.  This  review  is  interesting,  also,  as  it  shows  the 
slow  development  of  the  study  of  vegetable  proteins  and  how  the  several 
investigators  have  been  influenced  by  the  knowledge  of  the  animal  proteins 
prevailing  at  the  time  the  work  was  done.  In  carrying  out  his  investiga- 
tions of  these  proteins  the  writer  has  received  the  assistance  of  Messrs. 
Voorhees,  Campbell,  Harris,  and  Clapp,  for  which  he  wishes  here  to  make 
acknowledgment ;  but  especially  is  he  indebted  to  Prof.  S.  W.  Johnson, 
under  whose  direction  and  with  whose  advice  and  encouragement  this  work 
was  first  undertaken  in  the  laboratory  of  the  Connecticut  Agricultural 
Experiment  Station,  where  it  has  since  been  continued. 


REVIEW  OF  THE  LITERATURE. 

The  fact  that  gluten  can  be  obtained  from  wheat  flour  by  washing  with 
water  appears  to  have  been  first  published  by  Beccari.1 

That  alcohol  extracts  a  protein  substance  from  wheat  flour  was  first  stated 
by  Einhof, s  who  considered  this  to  be  the  same  as  the  gluten. 

Taddei3  found  that  gluten  consists  of  two  substances,  one  of  which  is 
soluble  in  alcohol,  which  he  named  "  gliadin,"  the  other  insoluble  in  alcohol, 
which  he  named  ' '  zymom. ' ' 

De  Saussure4  obtained  from  wheat  gluten  about  72  per  cent  of  plant- 
albumin  in  the  insoluble  form,  about  20  per  cent  of  plant-gelatin,  or,  as  he 
proposed  to  call  it,  "glutin,"  and  about  i  percent  of  mucin,  which  latter 
substance  he  considered  to  be  similar  to  the  mucin  described  by  Berzelius. 

Berzelius5  thought  that  the  alcoholic  extract  contained  another  protein 
substance,  which  he  called  "mucin,"  and  that  the  part  of  the  gluten  which 
was  insoluble  in  alcohol  was  so  similar  to  albumin  that  he  called  this 
' '  plant-albumin. ' ' 

Boussingault6  agreed  with  Einhof  that  the  part  of  the  gluten  that  was 
soluble  in  alcohol  was  the  same  as  the  entire  gluten  protein. 

1  Beccari.     Reference  to  this  publication  has,  for  many  years,  appeared  in  the  literature 
as  Comon.  Bonon.  I.  i,  p.  122.     It  should  be  De  Bononiensi  Scientiarum  et  Artium  Insti- 
tute atque  Academia  Commentarii,  1745,  ir,  part  i,  p.  122.     In  this  paper  Beccari  refers 
to  the  fact  that  in  1728  he  had  orally  communicated  to  the  Academy  the  previously  un- 
published fact  that  wheat  flour  can  be  separated  into  two  parts,  one  of  vegetable,  the 
other  of  animal  character.     The  substance  of  this  communication  was  published  in  the 
above-cited  paper  in  which  the  separation  of  gluten  from  wheat  flour,  by  washing  with 
water,  is  described. 

2  Einhof,  Journal  der  Chemie  von  Gehlen,  1805,  v,  p.  131. 

3  Taddei,  Annals  of  Philosophy,  1820,  xv,  p.  390. 

4  De  Saussure,  Schweiger's  Journal,  1833,  i,xix,  p.  188. 

'  Berzelius,  Lehrbuch  der  Chimie,  Auflage  3,  1837,  vi,  p.  453. 

6  Boussingault,  Annales  de  Chimie  et  de  Physique,  1838,  i«xv,  p.  30. 


REVIEW    OF   THE    LITERATURE.  7 

LJebig1  named  the  part  of  the  gluten  that  was  insoluble  in  alcohol  ' '  plant- 
fibrin,"  on  account  of  its  supposed  resemblance  to  blood-fibrin.  The  sub- 
stance soluble  in  alcohol  he  called  ' '  plant-gelatin ' '  and  considered  it  to  be 
a  compound  of  a  protein  with  an  organic  acid.  In  the  aqueous  extract  of 
the  flour  he  recognized  the  presence  of  albumin. 

Scherer2  prepared  plant-fibrin  by  dissolving  gluten  in  dilute  alkali,  filter- 
ing, neutralizing  with  acetic  acid,  and  extracting  the  precipitate  with  hot 
alcohol  and  then  with  ether. 

Bonchardat3  considered  that  wheat  gluten  contained  a  protein  soluble  in 
extremely  dilute  acids,  which  he  named  ' '  albutninose. ' ' 

Dumas  &  Cahours4  found  four  protein  substances  in  wheat  flour,  namely, 
plant-fibrin,  which  remained  after  extracting  gluten  with  alcohol ;  a  sub- 
stance which  they  considered  similar  to  casein,  which  was  deposited  by 
cooling  the  alcoholic  extract  ;  glutin,  which  was  obtained  by  concentrating 
and  cooling  the  alcoholic  extract,  and  albumin,  which  was  present  in  the 
aqueous  washings  of  the  gluten  and  was  coagulated  by  boiling.  The  plant- 
fibrin  they  considered  to  be  identical  with  blood- fibrin,  as  both  had  the  same 
ultimate  composition,  and  the  albumin  to  be  identical  with  egg-albumin  for 
the  same  reason. 

Mulder5  considered  the  plant-gelatin,  obtained  by  extracting  gluten  with 
alcohol,  to  be  a  compound  of  sulphur  with  "  protein,"  which  contained  the 
same  proportion  of  sulphur  as  blood-albumin. 

Von  Bibra6  recognized  three  proteins  in  gluten — plant-fibrin,  which 
formed  70.8  per  cent;  plant-gelatin,  16.2  per  cent;  and  plant-casein,  7.1 
per  cent.  In  the  water  used  for  washing  out  the  gluten  he  found  1.34  per 
cent  of  albumin. 

Giinsberg7  considered  that  Taddei's  view  that  there  were  only  two  pro- 
teins in  wheat  gluten  was  correct.  By  boiling  wheat  gluten  with  water  he 
obtained  five  preparations  which  separated  on  cooling  and  had  the  same 
ultimate  composition  as  has  been  established  for  gliadin.  By  treating 
gliadin  in  the  same  way  he  obtained  a  body  of  the  same  composition.  The 
substance  which  Giinsberg  thus  obtained  was  unquestionably  gliadin,  which 
is  sparingly  soluble  in  hot  water,  and  he  appears  to  have  been  the  first  to 
obtain  correct  analyses  of  this  protein. 

1 1/iebig,  Annalen  der  Chemie  und  Pharmacia,  1841,  xxxix,  p.  129. 

2Scherer,  Annalen  der  Chemie  und  Pharmacie,  1841,  xi,,  p.  7. 

8  Bonchardat,  ibid.,  1842,  xui,  p.  124. 

*  Dumas  &  Cahours,  Journal  fur  praktische  Chemie,  1843,  xxviu,  p.  398. 

5  Mulder,  Annalen  der  Chemie  und  Pharmacie,  1844,  ui,  p.  419. 

6  Von  Bibra,  Die  Getreidearten  und  das  Brod,  Nuremberg,  1860. 

7  Giinsberg,  Journal  fur  praktische  Chemie,  1862,  LXXXV,  p.  213. 


8 


THE    PROTEINS   OF   THE    WHEAT    KERNEL. 


Commaille1  recognized,  as  protein  constituents  of  flour,  sitosin,  soluble  in 
water  and  coagulable  by  heat ;  imesin,  soluble,  after  drying,  only  in  water 
containing  o.  i  per  cent  hydrochloric  acid  ;  sitesin,  soluble  in  o.  i  per  cent 
hydrochloric  acid ;  glutin,  nearly  insoluble  in  dilute  acid,  easily  in  strong  acid, 
forming  an  emulsion  with  alcohol,  which  is  separated  by  much  water  ;  and 
mucin,  which  dissolves  easily,  even  after  drying,  in  water  and  in  cold  80 
per  cent  alcohol. 

Ritthausen2  next  published  the  results  of  his  extensive  investigations  in  a 
volume  in  which  he  discussed  at  length  the  proteins  of  the  wheat  kernel. 
The  composition,  properties,  and  the  proportions  in  which  they  occur  in  the 
seed  were  given  in  detail,  and  also  the  evidence  which  he  considered  showed 
the  individuality  of  each.  He  recognized  five  proteins,  namely,  gluten-casein, 
gluten-fibrin,  plant-gelatin  or  gliadin,  and  mucedin,  as  constituents  of  the 
gluten,  and  also  albumin,  which  he  found  in  the  aqueous  extracts  of  the 
seed. 

The  gluten-casein  was  not  soluble  in  water,  very  slightly  soluble  in  dilute 
alcohol,  and  readily  soluble  in  very  dilute  acids  and  alkalis.  When  decom- 
posed by  boiling  with  sulphuric  acid  it  yielded  tyrosine,  leucine,  5.3  per 
cent  of  glutaminic  acid,  and  0.33  per  cent  of  aspartic  acid.  In  the  dry  gluten 
he  found  from  26  to  31.4  per  cent  of  gluten-casein,  which  he  considered  to 
be  minimal  quantities. 

Table  giving  results  as  ascertained  by  Ritthausen. 


Gluten- 
casein. 

Gluten- 
fibrin. 

Plant- 
gelatin. 

Mucedin. 

Albumin. 

Carbon  

P.Ct. 
S2.Q4 

P.Ct. 

"54.11 

P.Ct. 

S2.76 

P.Ct. 

54.11 

P.Ct. 

5V  12 

Hydrogen  .  . 

7.O4. 

7.18 

7.IO 

6.QO 

7.18 

Nitrogen  

17.14 

16.89 

18.01 

16.63 

I7.6O 

Sulphur  

O.O6 

I.OI 

0.85 

0.88 

I.SS 

Oxveen  .  . 

21.  Q2 

20.61 

21.  37 

21.48 

20.  s  5 

IOO.OO 

100.00 

100.00 

100.00 

IOO.OO 

The  gluten-fibrin  formed  that  fraction  of  the  alcohol- soluble  proteins 
which  was  soluble  in  the  strongest  alcohol  and  separated  from  a  hot  con- 
centrated solution  in  50  to  60  per  cent  alcohol  on  cooling.  Owing  to  the 
difficulty  encountered  in  separating  gluten-fibrin  from  the  other  alcohol- 
soluble  proteins,  it  was  impossible  to  determine  its  amount.  Usually  from 


1  Commaille,  Journal  de  Pharmacie,  1866  (4),  iv,  p.  108. 
'Ritthausen,  Die  Eiweisskorper,  etc.,  Bonn,  1872. 


Of   THE    LITERATURE.  9 

2  to  3  per  cent  of  the  gluten  was  obtained,  which  corresponds  to  0.25  to  0.35 
per  cent  of  the  flour.  The  actual  quantity  he  considered  to  be  much  more, 
and  that  in  different  varieties  of  wheat  the  proportion  of  gluten-fibrin  varied 
greatly. 

Ritthausen  described  gluten-fibrin  as  insoluble  in  water,  but  by  boiling 
with  water  it  was  decomposed  and  rendered  insoluble  in  alcohol.  In 
alcohol  of  30  to  70  per  cent  it  dissolved  readily  when  heated,  and  sepa- 
rated again  on  cooling,  more  completely  from  the  more  dilute  alcohol. 
From  dilute  solutions  on  concentration  and  from  concentrated  solutions  on 
cooling  the  gluten-fibrin  separated  on  the  surface  of  the  liquid  as  a  thick, 
soft  skin,  which  was  renewed  as  often  as  it  was  removed,  which  property 
Ritthausen  considered  distinguished  it  from  mucedin  and  plant-gelatin.  In 
cold  alcohol  of  80  to  90  per  cent  the  gluten-fibrin  was  soluble  to  a  consider- 
able degree.  Dilute  acids  and  alkalis  dissolved  this  protein  freely,  yielding 
solutions  from  which  it  was  precipitated  on  neutralizing  to  a  slight  acid 
reaction. 

The  composition  of  gluten-fibrin  is  shown  in  the  table  on  page  8. 

Plant-gelatin  or  gliadin  formed  the  fraction  of  the  alcohol-soluble  protein 
that  dissolved  freely  in  alcohol  of  60  to  70  per  cent.  The  solubility  of  this 
protein  decreased  rapidly  when  the  proportion  of  alcohol  to  water  fell  below 
or  above  this  strength.  It  was  very  slightly  soluble  in  cold  water,  more 
so  in  hot  water.  By  boiling  with  water  it  was  gradually  rendered  insol- 
uble in  alcohol.  Extremely  dilute  acids  and  alkalis  dissolved  plant-gelatin 
readily. 

The  amount  of  plant-gelatin  which  different  wheats  contain  was  not  deter- 
mined, owing  to  the  impossibility  of  separating  it  from  the  other  proteins. 
The  composition  of  this  protein  is  shown  in  the  preceding  table. 

Mucedin  formed  the  fraction  of  the  alcohol- soluble  proteins  which  was 
soluble  in  the  most  dilute  alcohol.  Except  for  its  greater  solubility  in  water 
and  in  very  dilute  alcohol,  mucedin  does  not  appear  to  differ  greatly  in  its 
properties  from  plant-gelatin.  Its  composition  is  given  in  the  table  on 
page  8. 

Mucedin  yielded  25  per  cent  of  glutaminic  acid  when  boiled  with  sul- 
phuric acid,  but  other  decomposition  products  were  not  determined.  Only 
a  very  small  quantity  of  mucedin  was  obtained  in  a  pure  state,  and  no 
estimate  of  its  total  amount  was  made.  The  relative  proportion,  however, 
Ritthausen  considered  to  vary  greatly  in  different  sorts  of  wheat. 

Albumin  was  obtained  by  heating  the  acidified  wash- waters  of  the  gluten. 
This,  however,  he  considered  as  possibly  derived  from  the  soluble  part  of 
the  gluten-proteins.  The  composition  of  this  albumin  is  given  in  the  table 
on  page  8. 


IO  THE    PROTEINS   OF   THE    WHEAT    KERNEL. 

Weyl1  was  the  first  to  recognize  the  presence  of  globulin  in  wheat  flour, 
and  states  that  besides  vegetable-vitellin,  vegetable-myosin,  which  coagu- 
lated at  55°  to  60°,  was  also  present. 

Weyl  &  Bischoff 2  considered  the  protein  matter  of  wheat  to  consist  chiefly 
of  a  myosin-like  globulin  which  they  called  vegetable-myosin,  and  that,  if 
so,  this  must  be  the  substance  from  which  gluten  is  derived,  for  other  pro- 
teins are  present  only  in  small  quantity.  Extraction  with  15  per  cent  salt 
solution  left  a  residue  from  which  they  obtained  no  gluten.  They  therefore 
considered  it  probable  that  the  gluten  forms  from  the  myosin  in  consequence 
of  a  ferment  action  similarly  to  the  formation  of  fibrin  from  fibrinogen. 
No  ferment,  however,  could  be  detected.  They  also  found  that  large 
amounts  of  sodium  chloride,  sodium  sulphate,  and  magnesium  sulphate 
hindered  the  formation  of  gluten  in  the  same  way  that  sodium  and  mag- 
nesium sulphates  hinder  the  formation  of  fibrin.  As  no  gluten  was  obtained 
from  flour  extracted  with  alcohol,  they  concluded  that  the  myosin  had  been 
coagulated.  By  warming  flour  48  to  96  hours  below  60°,  the  coagulation 
point  of  myosin,  no  gluten  was  obtained  from  the  meal  after  adding  a  little 
unwarmed  flour,  showing  that  the  gluten-forming  substance  had  been 
coagulated. 

Balland3  found  that  nearly  the  same  amount  of  gluten  was  formed  with 
water  at  2°,  15°,  and  60°,  and  therefore  concluded  that  no  ferment  action 
took  part  in  its  formation. 

According  to  Martin,4  alcohol  extracts  from  gluten  but  one  protein  sub- 
stance. This  is  soluble  in  hot  water,  but  insoluble  in  cold  ;  hence  is  insoluble 
phytalbumose.  The  residue  remaining  after  treatment  with  alcohol  is 
uncoagulated  protein,  soluble  in  dilute  acids  and  alkalis.  This  he  called 
"gluten-fibrin."  The  insoluble  phytalbumose  is  not  present,  as  such,  in 
flour,  since  direct  extraction  of  the  meal  with  75  per  cent  alcohol  removes 
no  protein.  Extraction  with  water  yields  less  globulin  and  soluble  albumose 
than  extraction  with  sodium  chloride  solution  of  10  to  15  per  cent.  Martin 
therefore  concluded  that  the  insoluble  phytalbumose  is  formed  from  the 
soluble  by  the  action  of  water,  the  gluten-fibrin  being  formed  by  a  similar 
action  of  water  on  the  globulin  ;  that  is,  conversion  into  an  albuminate. 
This  albuminate  and  the  insoluble  phytalbumose  together  constitute  gluten. 

W.  Johannsen5  believed  that  there  was  no  ferment  action  in  the  formation 
of  gluten.  Dough  was  obtained  by  grinding  dried  gluten  and  mixing  with 
starch,  and  also  by  mixing  moist  gluten  with  starch. 

1  Weyl,  Zeitscbrift  fur  physiologische  Chemie,  1877,  i,  p.  72. 

1  Weyl  &  Bischoff,  Berichte  der  deutschen  chemischen  Gesellschaft,  1880,  xiu,  p.  367. 

3  Balland,  Comptes  rendus  de  I'Acade'mie  des  Sciences,  1883,  cxv,  p.  202. 

4  Martin,  British  Medical  Journal,  1886,  II.  p.  104. 

5W.  Johannsen,  Annales  Agronomiques,  1888,  xiv,  p.  420. 


REVIEW  OF  THE  LITERATURE.  n 

Chittenden  &  Smith1  prepared  many  samples  of  gluten-casein  according  to 
Ritthausen's  method.  As  an  average  of  eight  analyses  they  found  the  fol- 
lowing composition  : 

p.  ct. 

Carbon 52.87 

Hydrogen 6.99 

Nitrogen 15.86 

Sulphur 1.17 

Oxygen 23. 1 1 


They  also  prepared  and  analyzed  the  various  products  of  peptic  digestion 
of  this  protein. 

Osborne  &  Voorhees2  investigated  the  number  and  character  of  the  proteins 
of  wheat,  but  as  their  results  are  given  in  detail  in  the  body  of  this  paper 
they  need  not  here  be  further  mentioned. 

O'Brien*  recognized  globulin,  proteose,  and  the  gluten-proteins  in  extracts 
of  wheat  flour.  The  protein-leucosin,  which  Osborne  &  Voorhees  considered 
to  be  an  albumin,  was  regarded  as  a  globulin  by  O'Brien,  since  it  is  precip- 
itated by  saturating  its  solutions  with  magnesium  sulphate. 

About  i  per  cent  of  the  flour  consists  of  proteins  soluble  in  saline  solu- 
tions and  coagulating  on  boiling.  Neither  ferment  action  nor  globulin  take 
part  in  gluten  formation.  Gluten  consists  of  zymom,  insoluble  in  alcohol, 
and  glian,  soluble  therein.  Glian  is  formed  by  hydration  of  the  protein  of 
the  flour  and  zymom  from  glian  by  further  hydration.  Glian  yields  myxon, 
glutine,  and  mucine,  which  are  not  constituents  of  glian,  but  derived  from  it. 

Frankfurt*  estimated  the  proportion  of  various  constituents  of  the  embryo 
of  wheat,  and  found  globulin  21.62  per  cent  and  albumose  13.62  per  cent. 

O'Brien5  stated  that  the  proteins  of  the  wheat  embryo  consist  of  globulins 
of  themyosin  type,  coagulating  at  55°,  soluble  in  dilute  solutions  of  sodium 
chloride  or  magnesium  sulphate  and  precipitated  by  excess  of  these  salts  ; 
globulins  of  the  vitellin  type,  coagulating  at  75°  to  78°,  and  soluble  in  dilute 
solution  of  sodium  chloride,  but  not  precipitated  by  an  excess ;  proteose 
and  albumin,  not  coagulating  below  80°,  soluble  in  sodium  chloride  solution, 
but  not  precipitated  by  an  excess,  nor  by  dialysis,  nor  by  carbonic  acid. 

Kjeldahl6  found  that  all  of  a  number  of  preparations  of  the  alcohol- soluble 
protein  made  from  wheat  flour  showed  an  almost  constant  content  of  about 
52  per  cent  of  carbon  and  17.25  per  cent  of  nitrogen,  and  when  dissolved  in 

1  Chitteuden  &  Smith,  Journal  of  Physiology,  1890,  xi,  p.  419. 

'*  Osborne  &  Voorhees,  American  Chemical  Journal,  1893,  xv,  p.  392. 

3  O'Brien,  Annals  of  Botany,  1895,  ix,  p.  171. 

*  Frankfurt,  Versuchs-Stationen,  1895,  xi^vn,  p.  449. 

5  O'Brien,  Annals  of  Botany,  1895,  ix,  p.  543. 

6  Kj Adahl,  Agricultur  chemischen  Centralblatt,  1896,  xxv,  p.  197. 


12  THE    PROTEINS    OF   THE    WHEAT    KERNEL. 

75  per  cent  alcohol  a  specific  rotation  of  — 92°.  This  rotation  was  so  con- 
stant, not  only  for  the  protein  from  different  sorts  of  wheat  from  different 
regions,  but  also  for  those  from  crops  of  four  different  years,  that  it  seemed 
to  Kjeldahl  that  wheat  flour  contained  only  one  single  protein  substance 
soluble  in  alcohol. 

Fleurent1  held  the  view  that  only  one  protein  substance  soluble  in  alcohol 
was  present  in  wheat  flour,  and  proposed  a  method  for  determining  the 
amount  of  gliadin  and  glutenin.  By  this  method  he  found  that  gluten  con- 
tained from  60  to  80  per  cent  of  gliadin  and  18  to  25  per  cent  of  glutenin, 
according  to  the  variety  of  the  wheat  from  which  it  was  obtained. 

Guthrie2  concluded  that  the  water-absorbing  power  of  wheat  flour  was 
greater  when  the  proportion  of  glutenin  to  gliadin  was  greater,  strong  flours 
being  those  relatively  rich  in  glutenin. 

Teller3  devised  methods  for  determining  the  relative  quantities  of  the 
different  proteins  in  wheat  flour  and  applied  them  to  flours  of  different 
origin  and  to  various  mill  products. 

Teller*  also  concluded  that  the  proteose  found  by  Osborne  &  Voorhees 
was  gliadin  that  had  been  dissolved  in  small  quantity  in  the  aqueous  extract. 
Osborne6  showed  that  this  was  erroneous  and  gave  the  reasons  why  he  had 
not  mistaken  gliadin  for  proteose. 

Morishima's6  investigations  led  him  to  believe  that  wheat  gluten  contained 
but  a  single  protein,  and  that  glutenin  and  gliadin  were  derivatives  of  one 
and  the  same  substance,  which  he  named  artolin. 

Teller7  determined  the  proportion  of  the  several  proteins  present  in  the 
wheat  kernel  on  many  consecutive  days  during  the  ripening  of  the  grain 
and  found  a  large  increase  of  gliadin  nitrogen  during  this  period,  together 
with  a  smaller  though  marked  decrease  of  the  glutenin  nitrogen  when  consid- 
ered in  proportion  to  the  whole  amount  of  nitrogen  present.  The  changes 
in  the  proportion  of  leucosin  and  globulin  nitrogen  were  less  marked  and 
more  irregular. 

Ritthausen8  again  asserted  his  belief  in  the  existence  of  three  distinct 
proteins  in  wheat  that  were  soluble  in  alcohol,  but  offered  no  new  evidence 
of  their  existence. 

1  Fleurent,  Comptes  rendus  de  1'Acad^mie  des  Sciences,  1896,  cxxrn,  p.  755. 

9  Guthrie,  Agricultural  Gazette  of  New  South  Wales,  September,  1896. 

3  Teller,  Arkansas  Agr.  Exp.  Sta.  Bull.  42,  part  2,  p.  81.     1896. 

*  Teller,  American  Chemical  Journal,  1897,  xix,  p.  65. 

6  Osborne,  ibid.,  1897,  xix,  p.  263. 

6  Morishima,  Archiv  fiir  experim.  Pathologie  und  Pharmakologie,  1898,  xu,  p.  348. 

T  Teller,  Arkansas  Agr.  Exp.  Sta.,  Bull.  53,  p.  53.     1898. 

8  Ritthausen,  Journal  fiir  praktische  Chemie,  1899,  Lix,  p.  474. 


REVIEW  OF  THE;  IJTERATURE.  13 

Snyder1  determined  the  amount  of  the  different  proteins  in  various  flours 
and  mill  products.  He  found  73.9  per  cent  of  the  total  protein  to  be 
gliadin  in  patent  flour  from  soft  winter  wheat  and  63.7  per  cent  in  that  from 
hard  winter  wheat.  He  concluded  that  the  protein  in  the  gluten  of  a  flour 
good  for  bread-making  consists  of  65  per  cent  of  gliadin  and  35  per  cent  of 
glutenin.  The  ratio  of  gliadin  to  glutenin  in  different  grades  of  flour  varies 
between  i  to  4  and  nearly  2  to  i  .  While  the  lower  grades  of  flour  contain 
more  protein  than  the  higher,  the  proportion  of  gliadin  to  glutenin  is  not 
such  as  to  produce  bread  of  the  best  physical  properties. 

Osborne  &  Campbell*  found  that  the  leucosin,  globulin,  and  proteose, 
obtained  in  very  small  quantity  from  the  entire  wheat  kernel,  together  con- 
stitute nearly  the  whole  of  the  protein  of  the  embryo,  and  that  gliadin  and 
glutenin,  which  are  the  principal  proteins  of  the  endosperm,  could  not  be 
obtained  from  the  embryo.  The  details  of  this  investigation  are  given  in 
full  in  subsequent  pages  of  this  paper. 

Kossel  &  Kutscher,3  following  Ritthausen's  directions,  prepared  the  pro- 
teins of  wheat  gluten  and  determined  the  proportion  of  basic  products  which 
they  yielded  on  decomposition  with  acids.  They  found  that  glutenin  was 
sharply  distinguished  from  the  protein  soluble  in  alcohol  by  the  fact  that  it 
yields  a  notable  quantity  of  lysine,  whereas  all  their  products  derived  from 
the  alcoholic  extract  of  gluten  yielded  none  of  this  diamino-acid.  They 
held  the  view,  advanced  by  Ritthausen,  that  in  gluten  there  are  three  pro- 
tein substances  soluble  in  alcohol.  Of  these  mucedin  yielded  3.13,  gliadin 
2.75,  and  gluten-fibrin  3.05  per  cent  of  arginine  and  0.43,  1.20,  and  1.53 
per  cent  respectively  of  histidine  ;  but  in  view  of  the  methods  employed 
for  the  determinations  of  these  bases  they  consider  these  differences  too 
small  to  justify  the  conclusion  that  these  are  distinct  protein  substances. 

Dennstedt4  decomposed  "  wheat  fibrin  "  by  boiling  with  baryta  and  found 
that  one-third  of  the  nitrogen  was  split  off  as  ammonia  and  one-fifth  of  the 
sulphur  as  sulphide  and  sulphate.  After  removing  the  barium  and  treating 
the  solution  with  lead  acetate  he  separated  proteoses,  which  he  analyzed. 

Osborne5  made  careful  determinations  of  total  sulphur  in  four  samples  of 
thoroughly  purified  gliadin  and  found  an  average  of  1.027  per  cent,  of  which 
0.619  per  cent  was  split  off  as  sulphide  by  boiling  with  caustic  alkali.  * 


,  Minnesota  Agr.  Exp.  Sta.,  Bull.  63.     1899. 

2  Osborne  &  Campbell.  Journal  American  Chemical  Society,  1899,  xxi,  p.  486. 

3  Kossel  &  Kutscher,  Zeitschrift  fur  physiologische  Chemie,  1901,  xxxi,  p.  165. 

4  Dennstedt,  Chemiker  Zeitung,  1901,  p.  5. 

5  Osborne,  Journal  American  Chemical  Society,  1902,  xxiv,  p.  140. 


14  THE    PROTEINS   OF   THE    WHEAT    KERNEL. 

Osborne  &  Harris,1  in  a  study  of  the  different  forms  of  binding  of  nitrogen 
in  proteins,  found  that  leucosin  of  wheat  yielded  1.16  per  cent  of  nitrogen 
as  ammonia  and  3.50  per  cent  of  nitrogen  in  basic  compounds,  precipitable 
by  phosphotungstic  acid  ;  that  the  globulin  yielded  1.42  per  cent  of  nitrogen 
as  ammonia  and  6.83  percent  of  basic  nitrogen;  that  gliadin  yielded  4.3 
per  cent  of  nitrogen  as  ammonia  and  1.09  per  cent  of  basic  nitrogen,  and 
that  glutenin  yielded  3.31  per  cent  of  nitrogen  as  ammonia  and  2.05  per  cent 
of  basic  nitrogen.  They  also  found2  that,  while  none  of  the  wheat  proteins 
yielded  any  furfurol  on  distillation  with  hydrochloric  acid,  glutenin  gave  a 
moderately  strong,  gliadin  a  strong,  and  leucosin  a  very  strong  reaction  with 
the  Molisch  test.  This  test  is  commonly  regarded  as  giving  evidence  of  a 
carbohydrate  complex  in  the  protein  molecule,  but  they  decided  that  other 
evidence  is  necessary  before  such  a  conclusion  is  justified. 

Osborne  &  Harris3  found  the  specific  rotation  of  gliadin  dissolved  in 
alcohol  of  80  per  cent  by  volume  to  be  —91.9°  and  —92.5°.  In  comparing 
the  tryptophane  reaction  of  many  proteins  these  same  authors  found4  that 
the  globulin  of  wheat  gave  only  a  slight  reaction,  gliadin  and  glutenin  one 
of  medium  intensity,  and  leucosin  the  strongest  reaction  of  all  the  proteins 
examined,  thus  indicating  the  relative  amounts  of  tryptophane  or  indol- 
amino-propionic  acid  which  these  proteins  yield  on  decomposition  with  acid. 

Kutscher5  determined  the  amount  of  tyrosine  and  glutaminic  acid  yielded 
by  the  proteins  of  wheat  gluten  when  decomposed  by  boiling  with  sulphuric 
acid.  In  glutenin  he  found  2.75,  in  gluten-fibrin  4.^-^  'Un  2.09,  and 

in  mucediu  2.35  per  cent  of  tyrosine  ;  in  glutenin  <j  .cen-fibrin  13.07, 

in  gliadin  18.54,  and  in  mucedin  19.81  per  cent  of  g  jiinic  acid. 

Naysmith6  found  that  gluten  contained  0.12  per  cent  of  phosphorus. 
Gliadin  extracted  by  70  per  cent  alcohol  from  gluten  and  separated  by 
evaporating  to  dryness  contained  0.29  per  cent  of  phosphorus,  but  when 
precipitated  from  the  alcoholic  extract  by  dilute  sodium  chloride  solution  it 
contained  only  0.19  per  cent. 

Glutenin  also  contained  o.  2 1  per  cent  of  phosphorus.  Both  these  proteins 
contained  iron.  Phosphorus  and  iron  are  not  constituents  of  the  molecules 
of  these  proteins,  but  are  derived  from  the  cell  nuclei. 

Although  gliadin  and  glutenin  contain  phosphorus,  they  are  not  nucleo- 
proteids.  Naysmith  also  concluded  that  no  ferment  action  occurred  in  the 
formation  of  gluten. 

1  Osborne  &  Harris,  Journal  American  Chemical  Society,  1903,  xxv,  p.  323. 

2  Ibid. ,  p.  474. 
3 Ibid.,  p.  844. 
*Ibid.,  p.  854. 

5  Kutscher,  Zeitschrift  fur  physiologische  Chetnie,  1903,  xxxvin,  p.  in. 

6  Naysmith,  Transactions  of  the  Canadian  Institute,  1903,  vil. 


OF  THE;  LITERATURE. 


The  composition  of  gliadin  and  glutenin  as  prepared  by  Naysmith  were 
as  follows  : 

Composition  of  gliadin  and  glutenin  as  prepared  by  Naysmith. 


Gliadin. 

Glutenin. 

Carbon    

P.ct. 
52.39 

P.ct. 
52.75 

Hydrogen  

6.84 

7.22 

Nitrogen       .  .        

17.47 

16.15 

Sulphur  

1.  12 

i.  06 

Ox  v  sen  .  , 

21.89 

22.58 

Phosphorus  

0.267 

0.215 

Iron         

0.034 

0.026 

IOO.OCO 

IOO.OOO 

Snyder1  proposed  a  method  for  determining  gliadin  in  wheat  flour  which 
was  based  on  the  optical  rotation  of  the  alcoholic  extract  of  a  definite 
quantity  of  the  flour.  The  results  of  this  method  agreed  closely  with  those 
obtained  by  determining  nitrogen  in  the  alcoholic  extract.  The  specific 
rotation  of  gliadin  calculated  from  the  mean  of  several  determinations  by 
the  above  method  is  («)D  =  —90°,  approximately. 

Chamberla/:"::.  ,1  f^the  method  of  determining  gliadin  and  glutenin  in 
wheat  flour  whic;  .  --ij^en  proposed  by  Fleurent  arid  modified  by  Manget, 
and  found  that  the  it,,,  arts  for  gliadin  were  too  high  and  for  glutenin  too 
low.  He  therefore  proposed  a  method  based  on  the  work  of  Snyder  and  of 
Osborne  &  Voorhees. 

Konig  &  Rintelen3  published  an  account  of  their  investigation  of  the  pro- 
teins of  wheat  gluten  and  conclude  with  Ritthausen  that  there  are  three 
soluble  in  alcohol.  Their  analyses  of  the  preparations  representing  these 
three  proteins  agreed  closely  with  those  of  Ritthausen  for  gliadin,  but 
differed  considerably  in  carbon  for  those  representing  gluten-fibrin  and 
mucedin. 

Osborne  &  Harris*  reviewed  the  work  that  had  been  done  on  the  alcohol- 
soluble  proteins  of  wheat  published  since  1893  and  gave  their  reasons  for 
adhering  to  the  views  of  Osborne  &  Voorhees,  that  only  one  such  protein 

1  Snyder,  Journal  American  Chemical  Society,  1904,  xxvi,  p.  263. 
'Chamberlain,  U.  S.  Dept.  of  Agriculture,  Bureau  of  Chemistry,  1904,  Bulletin  81. 
*  Konig  &  Rintelen,  Zeitschrift  f iir  Untersuchung  der  Nahrungs  und  Genussmittel, 
1904,  vin,  p.  401. 
4  Osborne  &  Harris,  American  Journal  of  Physiology,  1905,  xin,  p.  35. 


i6 


THE    PROTEINS    OE   THE    WHEAT    KERNEL. 


existed,  namely,  gliadin,  the  gluten-fibrin  and  mucedin  being,  in  their 
opinion,  impure  preparations  of  gliadin.  This  opinion  was  supported  by 
further  experimental  evidence  and  by  quantitative  determinations  of  the 
glutaminic  acid  yielded  by  hydrolyzing  different  fractional  preparations  of 
gliadin,  some  of  which,  according  to  Ritthausen's  statements,  should  have 
contained  the  gluten-fibrin.  The  amount  of  glutaminic  acid  found  in  the 
different  fractions  was  essentially  the  same. 

The  glutaminic  acid  thus  obtained  from  gliadin  was  37.3  per  cent,  which 
is  more  than  that  from  any  protein  substance  yet  examined. 

Abderhalden  &  Samuely1  determined  the  amount  of  the  various  primary 
decomposition  products  yielded  by  gliadin  and  found — 


p.  ct. 

Glycocoll 0.68 

Alanine 2.66 

Ammo  valeriatiic  acid o.  33 

Q-proline 2.40 

Leucine 6.00 

Glutaminic  acid 27.60 


P.  ct. 

Asparticacid 1.24 

Phenylalanine 2  60 

Serine o.  12 

Tyrosine 2.37 

Tryptophane,  about i  .00 


The  gliadin  used  for  this  analysis  yielded  12  per  cent  of  humus,  which, 
together  with  moisture  and  ash,  were  deducted  in  calculating  the  above 
percentages. 

Mathewson2  determined  the  specific  rotation  of  gliadin  in  various  organic 
solvents  with  the  following  results  : 


—  91  95 

—  96.66 


Methyl  alcohol,  70  per  cent  (a)  -^_ —  95.65 

Ethyl  alcohol,  70 
Ethyl  alcohol,  60 
Ethyl  alcohol,  50 

Propyl  alcohol,  60             '                     — 101. 10 

Phenol,  70 

Phenol,  anhydrous — 131-77 

Paracresol — 121.00 

Benzyl  alcohol —  53. 10 

Glacial  acetic  acid —  78.60 

Osborne  &  Harris*  described  the  preparation  of  large  quantities  of  the 
wheat  proteins  to  be  used  for  the  quantitative  determination  of  the  products 
of  hydrolysis.  These  determinations  were  made  by  Osborne  &  Clapp.4  The 
results  described  in  these  papers  are  given  in  full  later  in  this  publication. 

1  Abderhalden  &  Samuely,  Zeitschrift  fur  physiologische  Chemie,  1905,  xi,vi,  p.  193. 

2  Mathewson,  Journal  American  Chemical  Society,  1906,  xxvm,  p.  1482. 

3  Osborne  &  Harris,  American  Jourpal  of  Physiology,  1906,  xvn,  p.  223. 

4  Osborne  &  Clapp,  ibid.,  p.  231. 


EXPERIMENTAL.  17 

EXPERIMENTAL  PART. 

Although  positive  evidence  of  the  chemical  individuality  of  protein  sub- 
stances can  not  yet  be  obtained,  there  is  no  question  that  protein  preparations 
can  be  isolated  from  seeds  and  animal  tissues  which,  beyond  doubt,  represent 
distinctly  different  substances.  Thus  five  unquestionably  different  forms 
of  protein,  differing  in  composition,  solubility,  and  physical  characters,  can 
be  isolated  from  the  wheat  kernel.  Whether  each  of  these  is  itself  a  chem- 
ical individual  or  a  mixture  of  two  or  more  very  similar  substances  can  not 
at  present  be  asserted.  All  that  can  be  said  is  that  it  has  not  yet  been  pos- 
sible to  separate  them  into  fractions,  the  properties  of  which  indicate  a 
mixture. 

Owing  to  the  extreme  sensitiveness  of  proteins  to  the  action  of  acids, 
alkalis,  and  salts,  the  minor  differences  in  solubility  are  not  to  be  depended 
upon  as  a  basis  for  characterizing  individual  proteins.  Thus  the  protein 
edestin,  which  in  pure  water  is  entirely  insoluble,  in  the  presence  of  a  slight 
amount  of  acid  is  freely  soluble  therein.  The  addition  of  a  small  quantity 
of  a  neutral  salt  throws  the  edestin  out  of  this  acid  solution,  while  a  larger 
quantity  of  salt  at  once  redissolves  it. 

Such  differences  in  solubility  have  nothing  to  do  with  the  protein  mole- 
cule proper,  but  depend  on  the  formation  of  protein  salts,  the  solubility  of 
which  is  different  from  that  of  the  free  protein  itself.  As  the  formation  of 
such  protein  salts  depends  on  conditions  that  in  most  cases  can  not  be  taken 
into  account,  such  differences  in  solubility  can  not  be  made  a  basis  for  char- 
acterizing the  different  individual  proteins. 

We  are  therefore  limited,  in  dealing  with  such  problems,  to  the  more  marked 
differences  in  solubility,  such  as  that  in  alcohol,  strong  saline  solutions,  or 
alkalis,  and  to  constant  ultimate  composition  of  successive  fractional  precip- 
itations. Thus,  when  proteins  have  been  separated  into  fractions  which 
have  the  same  composition,  general  solubility,  and  physical  properties,  we 
are  not  justified  in  concluding  that  we  have  in  hand  a  single  individual  pro- 
tein. All  we  can  conclude  is  that  we  have  reached  the  limit  of  separation 
attainable  with  the  means  now  available,  and  that  for  the  present  we  must 
accept  such  products  as  the  simplest  units  with  which  we  can  now  deal  and 
which  for  the  present  must  serve  as  our  basis  for  further  study.  If,  on  the 
other  hand,  protein  preparations,  characterized  in  the  manner  above  de- 
scribed, show  distinct  and  constant  differences  from  one  another,  we  are 
justified  in  considering  them  to  be  differ£h^substances. 

That  the  wheat  kernel  contains  at  ^east  five  such  distinct  protein  sub- 
stances will  be  shown  in  the  following  pages. 


J8  THE  PROTEINS  OF  THE;  WHEAT  KERNEL. 

THE  AQUEOUS   EXTRACT   OF  WHEAT   FLOUR. 

The  water-extract  of  wheat  flour  is  of  a  straw-yellow  color,  becoming  red- 
brown  on  standing,  and  has  a  very  slight  acid  reaction  towards  litmus. 

Saturated  with  ammonium  sulphate,  a  bulky  precipitate  forms,  which  on 
standing  contracts,  showing  the  solution  to  contain  but  a  small  amount  of 
protein  matter.  After  24  hours  this  precipitate  can  be  completely  dissolved 
in  water,  giving  no  evidence  of  the  formation  of  insoluble  derivatives.  Sat- 
uration with  sodium  chloride  gives  a  small  precipitate.  Acetic  acid  in  the 
cold  solution  gives  no  precipitate  until  sodium  chloride  is  added. 

On  slowly  heating,  the  solution  becomes  turbid  at  48°  and  yields  a  floccu- 
lent  coagulum  at  52°.  After  heating  to  65°  for  some  time  and  filtering,  the 
solution  becomes  turbid  again  at  73°,  flocks  forming  in  very  small  amount 
at  82°.  No  more  separation  occurs  on  further  heating  the  extract  even  to 
boiling.  The  addition  of  a  little  acetic  acid  and  sodium  chloride  gives  a 
small  precipitate.  The  body  coagulating  at  52°  forms  the  greater  part  of 
the  protein  in  solution.  The  complete  coagulation  of  this  protein  is  accom- 
plished with  difficulty,  prolonged  heating  at  65°  being  necessary  to  cause  it 
to  separate  completely.  The  addition  of  sodium  chloride  greatly  facilitates 
the  final  coagulation.  The  temperature  at  which  the  flocculent  coagulum 
separates  depends  upon  the  rate  of  heating.  Unless  the  solution  is  heated 
very  slowly,  the  point  at  which  flocculation  occurs  is  much  above  52°. 

When  the  sodium-chloride  extract  of  the  wheat  flour  is  saturated  with 
ammonium  sulphate  and  the  precipitate  redissolved,  and  its  solution  dialyzed 
until  all  of  the  globulin  has  separated,  the  solution,  when  slowly  heated  in 
a  double  water-bath,  becomes  turbid  at  48°  and  flocks  separate  at  55°.  After 
heating  for  some  time  at  65°  and  filtering  off  the  coagulum,  the  solution, 
when  again  heated,  becomes  turbid  at  70°  and  a  very  minute  amount  of 
flocculent  coagulum  forms  at  80°.  Boiling  the  solution  after  filtering  gives 
no  more  precipitate,  and  nothing  is  obtained  by  adding  a  little  salt  and  acetic 
acid.  If  the  amount  of  salt  is  increased  and  acetic  acid  added,  a  precipitate 
results.  Kqual  volumes  of  a  solution  so  prepared  were  treated  with  20  per 
cent  of  sodium  chloride  and  a  little  acetic  acid.  To  the  first  the  salt  and 
acid  were  added  directly,  to  the  second  after  heating  to  65°  and  filtering  off 
the  coagulum,  and  to  the  third  after  heating  to  95°  and  likewise  filtering. 
The  first  portion  gave  the  most  precipitate,  the  last  the  least,  showing  that  the 
coagulable  proteins  are  thus  precipitated  by  salt  and  acid.  The  filtrate  from 
the  first  portion  when  neutralized  and  boiled  gave  no  precipitate,  indicating 
that  the  separation  of  the  albumin  was  complete. 

The  above  solution,  freed  from  globulins  by  dialysis,  gave  a  precipitate 
on  saturation  with  sodium  chloride,  the  filtrate  from  which  became  turbid 


EXPERIMENTAL.  19 

when  heated  to  43°,  flocculent  at  56°,  and  no  more  precipitation  on  further 
heating,  showing  that  either  a  higher-coagulating  protein  was  thus  removed 
or  that  coagulation  of  the  albumin  was  more  complete  in  the  strong  saline 
solution  at  the  lower  temperature.  This  dialyzed  solution  likewise  gave 
a  considerable  precipitate  with  nitric  acid.  On  heating,  a  part  remained 
insoluble,  and  on  filtering  this  off,  the  filtrate  gave  a  precipitate  on  cooling, 
which  dissolved  again  on  heating  and  reappeared  as  often  as  the  solution 
was  cooled.  The  filtrate  from  the  salt  and  acid  precipitate  did  not  give 
this  reaction,  but  the  solution  of  the  precipitate  in  water  gave  it  very 
strongly. 

This  reaction  is  characteristic  of  some  proteoses,  and  shows  that  the  salt 
and  acid  precipitate  contains  a  proteose  together  with  the  albumins.  This 
proteose  is  likewise  precipitated  by  saturating  the  extract  with  salt,  for 
on  dissolving  the  precipitate  so  produced  and  separating  the  albumin  con- 
tained in  it  by  coagulation  the  filtrate  gave  a  strong  red  biuret  reaction,  and 
a  precipitate  with  nitric  acid,  which  dissolved  on  warming  and  precipitated 
again  on  cooling.  The  filtrate  from  the  precipitate  caused  by  saturation 
with  salt  gives  no  reaction  with  nitric  acid,  showing  that  the  proteose  is  thus 
completely  precipitated. 

In  order  to  be  sure  that  the  coagulable  protein,  which  was  apparently  an 
albumin,  was  not  a  globulin  held  in  solution  -by  the  small  amount  of  salts 
contained  in  the  river-water  used  for  dialysis,  as  was  suggested  by  its  partial 
precipitation  by  saturation  with  sodium  chloride,  the  following  experiment 
was  tried  :  250  cc.  of  a  strong  aqueous  extract  of  winter-wheat  meal  was 
dialyzed  in  running  distilled  water  for  48  hours.  A  small  precipitate  was 
then  filtered  off,  the  clear  solution  returned  to  the  dialyzer,  and  the  process 
continued  for  five  days  longer.  No  more  substance  separated.  The  entire 
solution,  which  was  still  found  to  coagulate  at  54°,  was  then  evaporated  to 
dryness,  the  considerable  protein  residue  burned  off,  and  the  total  mineral 
matter  found  to  weigh  only  0.0008  gram,  thus  proving  the  protein  to  be  an 
albumin. 

We  are  able,  then,  to  recognize  two  distinct  protein  substances  soluble  in 
pure  water,  namely,  a  coagulable  albumin  and  a  proteose.  As  it  was  found 
that  the  protein  removed  from  the  flour  by  treatment  with  alcohol  was  to  a 
slight  extent  soluble  in  pure  water,  it  might  be  thought  that  one  of  these 
bodies  was  identical  with  this  alcohol-soluble  protein.  Its  identity  with  the 
albumin  is  excluded  by  the  fact  that  the  latter  is  precipitated  by  heat,  and 
with  the  proteose  by  the  fact  that  the  alcohol-soluble  proteose  gives  a  pre- 
cipitate with  hydrochloric  acid  when  it  is  dissolved  in  distilled  water  and  also 
with  a  little  sodium  chloride,  which  the  proteose  does  not. 


20 


THE;  PROTEINS  OF  THE  WHEAT  KERNEL. 


THE  ALBUMIN   OF  WHEAT   FLOUR — LEUCOSIN. 

A  quantity  of  wheat  flour  was  extracted  with  10  per  cent  sodium-chloride 
solution,  the  extract  saturated  with  ammonium  sulphate,  the  precipitated 
protein  dissolved  in  sodium-chloride  brine,  and  the  clear  solution  dialyzed 
until  all  the  globulin  had  been  precipitated.  The  filtered  solution  was  then 
heated  to  60°  in  a  water-bath  kept  between  60°  and  65°.  After  an  hour  the 
precipitate  was  filtered  off,  washed  thoroughly  with  water,  alcohol,  absolute 
alcohol  and  ether,  and  dried.  Before  drying,  the  coagulum  was  a  white, 
voluminous,  semisolid  mass,  which,  when  completely  dried  over  sulphuric 
acid,  became  dense  and  horny.  This  preparation,  i,  after  drying  at  110°, 
was  found  to  have  the  composition  shown  in  the  following  table  : 

Preparations  i  and  2. 


Preparation  I. 

Preparation  2. 

I. 

II. 

Aver- 
age. 

Ash- 
free. 

I. 

II. 

Aver- 
age. 

Ash- 
free. 

Carbon  

P.ct. 

53-04 
6.74 
16.86 
1.27 

P.ct. 

53-28 
6.89 
16.95 

P.ct. 
53-16 
6.82 
16.91 
1.27 

P.ct. 

53-27 
6.83 

16.95 
1.27 
21.68 

P.ct. 

52.91 

6.86 

16.93 
1.28 

P.ct. 

52.98 
6.74 
17.01 
I-3I 

P.ct. 
52.95 
6.80 
16.97 
1.30 

P.ct. 

53.06 
6.82 
17.01 
1.30 

21.  8l 

Hydrogen  .... 
Nitrogen  
Sulphur  

Ash  

O.22 

0.22 



100.00 

Another  lot  of  10,000  grams  of  flour  was  extracted  with  10  per  cent 
sodium  chloride  solution  and  treated  in  the  same  way  as  the  extract  last 
described.  The  albumin  obtained  weighed  6.4  grams  and  had  the  composi- 
tion given  in  the  above  table,  preparation  2. 

The  filtrate  from  preparation  2  was  then  heated  to  75°  and  0.65  gram 
of  coagulum  obtained,  which  on  analysis  gave  the  results  shown  in  the  table 
on  page  21,  preparation  3. 

Another  preparation  was  made  from  the  same  flour  by  extracting  10,000 
grams  with  10  per  cent  sodium  chloride  solution,  filtering  the  extract,  and 
dialyzing  at  once.  In  this  case  the  precipitation  of  the  proteins  with  ammo- 
nium sulphate  was  omitted.  After  complete  dialysis  the  solution  was  filtered 
from  the  globulin  which  had  separated,  and  the  solution  heated  at  once  to 
90° — a  temperature  high  enough  to  precipitate  all  of  the  albumin. 

Dried  over  sulphuric  acid,  this  preparation,  4,  which  in  all  respects  resem- 
bled i  and  2,  weighed  16.5  grams  and  when  analyzed  gave  the  results  shown 
in  the  table  at  the  top  of  page  2 1 . 


EXPERIMENTAL. 
Preparations  j,  4,  and  5. 


21 


Carbon  

Preparation  3. 

Preparation  4. 

Preparation  5. 

I. 

Ash-free. 

I. 

II. 

Average. 

Ash-free. 

I. 

Ash-free. 

P.  ct. 

P.ct. 

P.ct. 
S2.86 

P.  ct. 

P.ct. 
52.86 
6.85 
16.21 
1.  20 

P.ct. 
53-02 
6.87 
16.26 
i.  20 
22.65 

P.ct. 
52.36 
6.80 
16.62 
1-34 

P.ct. 
52.71 
6.85 
16.73 

i-34 
22.37 

Hydrogen  

6.85 

Nitrogen  

16.91 

16.94 

16.21 
i.  20 

16.20 

Sulphur  

Oxygen.  .  . 

Ash  

0.18 



0.32 



0.32 

0.67 

100  00 

IOO.OO 

Another  preparation  of  albumin  was  made  by  extracting  with  10  per  cent 
sodium  chloride  solution  2000  grams  of  so-called  ' '  shorts ' '  from  the  spring- 
wheat  flour.  This  substance  consisted  chiefly  of  particles  of  the  outer  coats 
of  the  seed  to  which  more  or  less  of  the  adjacent  embryo  and  endosperm 
adhered.  After  three  hours  the  extract  was  strained  through  a  coarse  cloth 
and  squeezed  out  from  the  residue  in  a  screw-press.  After  the  starch  had 
settled,  the  nearly  clear  extract,  which  had  a  deep  red-brown  color,  was 
siphoned  off  and  saturated  with  ammonium  sulphate.  The  precipitate  thus 
produced  was  filtered  off  and  dissolved  in  10  per  cent  sodium-chloride  brine. 
The  resulting  solution,  filtered  from  all  the  insoluble  matter,  was  then 
dialyzed  free  from  chlorides,  the  precipitated  globulins  filtered  off,  and  the 
albumin  contained  in  the  solution  separated  by  heating  to  65°.  This 
preparation,  5,  had  the  composition  shown  in  the  preceding  table. 

TABLE  i. — Summary  of  analyses  of  coagulated  wheat  albumin. 


i. 

2. 

3- 

4- 

5- 

Average. 

P.ct. 
•^.27 

P.ct. 
5^.06 

P.ct. 

P.ct. 

C7.O2 

P.ct. 

C2.7I 

P.ct. 

C1   O2 

Hydrogen  

6.8t 

6.82 

6.87 

685 

6  84 

Nitrogen  

i6.Q5 

17.  OI 

16  od 

16  26 

16  8^ 

16  80 

1.27 

I.^O 

1.  20 

i  \k 

i  28 

21.68 

21.81 

22.6=5 

22.27 

22.06 

IOO.OO 

IOO.OO 

IOO.OO 

IOO.OO 

IOO.OO 

The  agreement  of  the  figures  in  table  i  is  satisfactory,  with  the  exception 
of  the  nitrogen  in  4.  The  accuracy  of  this  analysis  in  this  respect,  however, 
can  not  be  doubted,  as  four  determinations  of  this  element  were  made,  all 
of  which  agreed  closely.  As  this  preparation  was  separated  at  a  higher 
temperature  than  any  of  the  others,  it  is  possible  that  it  had  in  consequence 
lost  some  of  its  nitrogen. 


22  THE)    PROTEINS    OF   THE    WHEAT    KERNEL. 

THE   AQUEOUS   EXTRACT   OF  THE   WHEAT  EMBRYO. 

The  embryo  flour,  when  treated  with  water,  yields  a  gummy  mass,  from 
which  a  clear  extract  is  secured  with  difficulty.  From  500  grams  of  meal 
an  extract  was  obtained  with  2000  cc.  of  water,  of  which  1400  cc.  could  be 
filtered  clear.  This  extract  was  neutral  to  litmus,  alkaline  to  lacmoid,  and 
so  acid  to  phenolphthalein  that  19  cc.  of  decinormal  alkali  were  required  to 
neutralize  100  cc.  of  it  to  this  indicator. 

When  a  freshly  prepared  dilute  aqueous  extract  of  the  recently  ground 
wheat  germs  is  heated  in  a  water-bath,  no  coagulation  occurs,  the  solution 
becoming  slightly  opalescent.  If  a  more  concentrated  extract,  such  as  may 
be  obtained  by  treating  one  part  of  meal  with  five  parts  of  water,  is  thus 
heated,  the  entire  solution  solidifies  to  a  firm,  opaque  jelly,  free  from  visible 
particles.  If  to  either  of  these  solutions  a  very  little  hydrochloric  acid 
is  added  previous  to  heating,  an  abundant  flocculent  coagulum  separates  on 
heating. 

After  standing  a  while  the  aqueous  extract  becomes  gradually  acid  to 
litmus,  so  that  when  heated  slowly  it  becomes  turbid  at  about  50°  and  a 
large  flocculent  coagulum  separates  at  55°.  Heated  to  65°  for  some  time 
and  filtered,  a  second  coagulum  may  be  obtained  on  raising  the  heat  from 
65°  to  1 00°.  The  amount  of  this  second  coagulum  is  about  one-third  that 
of  the  first. 

The  coagulated  protein  is  dissolved  by  0.5  per  cent  potassium-hydroxide 
solution,  but  not  perceptibly  by  0.4  per  cent  hydrochloric  acid  solution, 
unless  the  latter  is  heated,  when  a  clear  transparent  jelly  is  formed. 

Freed  from  coagulable  protein,  the  aqueous  extract  still  contains  a  rela- 
tively large  amount  of  substance  which  has  the  reactions  of  proteose. 

When  the  concentrated  aqueous  extract  is  poured  into  a  large  volume  of 
distilled  water,  a  turbidity  forms  at  first,  which  mostly  disappears  after 
shaking,  indicating  the  absence  of  a  notable  quantity  of  globulin  held  in 
solution  by  the  salts  dissolved  from  the  meal. 

Saturation  of  the  extracts  with  sodium  chloride  gives  a  considerable  pre- 
cipitate, only  a  small  part  of  which  can  be  redissolved  in  dilute  sodium- 
chloride  solution.  When  this  dissolved  part  is  precipitated  by  again  satu- 
rating with  sodium  chloride,  it  also  is  converted,  to  a  large  extent,  into  an 
insoluble  form  ;  the  part  still  remaining  in  solution  is  precipitated,  like  a 
globulin,  by  dialysis. 

When  the  solution,  saturated  with  sodium  chloride,  is  filtered  and  the 
diluted  filtrate  saturated  with  ammonium  sulphate,  a  part  of  the  precipitate 
produced,  when  redissolved  in  water,  is  thrown  out  of  solution  by  saturating 
with  sodium  chloride,  though  before  precipitation  with  ammonium  sulphate 
it  was  soluble  in  saturated  sodium-chloride  solution. 


EXPERIMENTAL,.  23 

These  reactions  show  that  changes  occur  which  involve  the  albumin  coag- 
ulating at  55°,  for  after  freeing  the  extract  from  all  protein  precipitable  by 
saturating  with  sodium  chloride  or  by  dialysis  there  remains  in  solution  only 
a  small  proportion  of  this  albumin. 

Thus  an  aqueous  extract  corresponding  to  666  grams  of  germ  meal,  when 
heated  to  65°,  yielded  62  grams  of  coagulum,  or  9.3  per  cent ;  a  similar  ex- 
tract on  dialysis  deposited  9.2  per  cent ;  only  0.87  per  cent  of  coagulable  and 
2.0  per  cent  of  uncoagulable  protein  remaining  in  solution.  The  precipitate, 
produced  by  dialysis,  was  but  slightly  soluble  in  sodium  chloride  solution, 
having  become  largely  coagulated.  From  these  facts  it  is  clear  that  one  and 
the  same  protein  substance  gives  rise  to  these  apparently  different  protein 
bodies,  and  consequently  the  substance  which  O'Brien  considered  to  be  a 
globulin  of  the  myosin  type  and  an  albumin,  coagulating  at  80°,  are  in  fact 
derivatives  of  the  albumin,  which  coagulates  mostly  at  65°. 

These  changes  are  due  to  a  slow  development  of  acid  in  the  extract,  which 
not  only  brings  about  hydroly tic  changes  in  the  protein,  but  may  also  lead  to 
the  formation  of  different  compounds  between  the  protein  and  the  various 
acids  contained  in  the  extract,  and  so  give  rise  to  chemically  different  sub- 
stances. Such  a  development  of  acid  takes  place  rapidly  in  muscle  plasma, 
under  the  influence  of  which  quite  similar  changes  in  the  proteins  there 
present  can  be  observed. 

Why  Frankfurt  overlooked  albumin,  present  in  such  large  proportion  in 
the  aqueous  extract,  is  not  easily  understood,  unless,  before  heating  his  solu- 
tions, he  either  added  no  acid  or  so  much  that  he  converted  this  substance 
into  an  uncoagulable  acid  compound. 

Hydrochloric  acid  added  to  the  extract  in  very  small  quantity  causes  a 
flocculent  coagulum  to  separate  on  heating,  while  a  slightly  larger  quantity, 
added  before  heating,  entirely  prevents  the  formation  of  this  coagulum. 
Acetic  acid  and  nitric  acid  give  precipitates  in  the  extracts  which  are  not 
soluble  in  a  reasonable  excess  of  either  of  these  acids. 

THE  ALBUMIN  OF  THE  WHEAT  EMBRYO. 

In  order  to  determine  definitely  the  relations  of  these  variously  obtained 
substances,  a  large  number  of  fractional  precipitations  have  been  made  under 
quite  different  conditions,  an  account  of  which  is  now  given  • 

An  extract  was  made  by  treating  700  grams  of  germ  meal  with  5  liters  of 
water,  straining  through  bolting-cloth  and  filtering  the  fluid  perfectly  clear. 
A  portion  of  it  was  at  once  heated  for  i  hour  in  a  water-bath  at  60°,  and  the 
large  coagulum  produced  gave  24  grams  of  preparation  6. 

Another  preparation  was  made  by  heating  in  a  water-bath  at  65°  2000  cc. 
of  a  clear  aqueous  extract,  obtained  by  treating  3000  grams  of  the  germ  meal 


24  THE;  PROTEINS  OF  THE  WHEAT  KERNEL. 

with  9  liters  of  water.  The  coagulum  produced,  weighing  62  grams,  formed 
more  than  g  per  cent  of  the  oil-free  germ  meal.  This  is  preparation  7. 

Another  aqueous  extract  was  heated  at  65°  until  all  the  protein  coagu- 
lable  at  this  temperature  had  separated.  The  coagulum  produced,  when 
washed  with  hot  water  and  alcohol,  was  dried  over  sulphuric  acid  and 
found  to  weigh  16.68  grams.  The  filtrate  from  this  coagulum,  heated  in  a 
boiling  water-bath,  yielded  a  second  coagulum  which  formed  preparation  8, 
weighing  4.9  grams. 

A  portion  of  the  extract  which  yielded  preparation  6  was  saturated  with 
ammonium  sulphate;  the  resulting  precipitate  was  dissolved  as  far  as  possi- 
ble in  water,  its  solution  filtered  clear  and  dialyzed  for  4  days.  During 
this  time  a  considerable  precipitate  formed  that,  when  filtered  out,  was 
found  to  be  insoluble  in  salt  solution.  The  solution,  filtered  from  that  sub- 
stance and  dialyzed  in  running  water  until  nothing  more  separated,  was 
filtered  and  heated  at  60°,  which  caused  a  coagulum.  This  coagulum 
weighed  7.1  grams  and  made  preparation  9. 

When  2000  cc.  of  an  extract  of  650  grams  of  wheat-germ  meal  was  dia- 
lyzed 4  days,  a  dense  turbidity  formed,  due  to  a  globulin,  since  it  dissolved 
on  adding  sodium  chloride.  Passing  carbon  dioxide  gas  through  the  dialyz- 
ing  solution  seemed  to  increase  the  turbidity,  but  effected  no  definite  separa- 
tion. As  it  was  found  that  10  cc.  of  decinormal  hydrochloric  acid  per  100  cc. 
of  the  extract  caused  a  separable  precipitate,  this  proportion  was  added  and 
the  resulting  flocculent  precipitate  brought  into  solution  again  by  adding 
sodium  chloride.  The  clear  solution  was  then  dialyzed  for  2  days  in  run- 
ning water  and  filtered  from  an  amorphous  precipitate,  which  was  treated  as 
later  described  on  page  44. 

The  filtrate  from  this  precipitate  was  further  dialyzed  for  3  days  more  in 
running  water  and  then,  as  nothing  separated,  for  4  days  more  in  alcohol. 
The  precipitate  which  resulted  was  dried  over  sulphuric  acid,  exhausted 
with  water,  in  order  to  remove  all  uncoagulated  proteins,  as  well  as  other 
soluble  substances,  dehydrated  with  absolute  alcohol,  again  dried  and 
weighed,  yielding  12  grams  of  preparation  10. 

Another  aqueous  extract  was  saturated  with  pure  sodium  chloride,  the 
abundant  precipitate  filtered  out,  treated  with  dilute  brine,  and  the  resulting 
solution  filtered  from  a  relatively  considerable  quantity  of  insoluble  matter. 
This  filtrate  was  saturated  with  sodium  chloride,  a  second  precipitate  filtered 
out,  and  likewise  treated  with  dilute  sodium-chloride  solution.  The  insol- 
uble portion  was  removed  by  filtration  and  the  clear  filtrate  dialyzed.  The 
small  precipitate  separated  by  dialysis  when  washed  and  dried,  weighed  4.8 
grams  and  formed  preparation  1 1 . 


EXPERIMENTAL.  25 

The  filtrate  from  the  first  precipitation  of  the  substance  of  preparation  1 1 , 
caused  by  saturating  its  solution  with  sodium  chloride,  as  described  above, 
was  diluted  with  water  and  saturated  with  ammonium  sulphate.  The  pre- 
cipitate which  resulted  was  dissolved  in  water  and  its  solution  precipitated 
by  saturating  with  sodium  chloride.  Although  this  substance  had  previ- 
ously been  soluble  in  saturated  brine,  after  precipitation  with  ammonium 
sulphate  it  was  found  to  be  nearly  all  insoluble  therein,  so  that  almost  com- 
plete precipitation  resulted  on  again  saturating  with  sodium  chloride.  The 
precipitate  so  produced  was  filtered  out,  dissolved  in  dilute  sodium  chloride 
solution,  and  reprecipitated  by  dialysis.  We  thus  secured  7.6  grams  of 
preparation  12. 

By  saturating  another  aqueous  extract  of  germ  meal  with  sodium  chloride 
a  very  large  quantity  of  protein  was  separated,  which  was  filtered  out,  ex- 
hausted with  dilute  sodium  chloride  solution,  and  the  insoluble  part  washed 
thoroughly  with  water  and  alcohol.  This  preparation,  13,  weighed  17 
grams. 

The  filtrate  and  saline  washings  from  preparation  13  were  united  and  again 
saturated  with  sodium  chloride  and  yielded  a  small  precipitate,  which,  when 
dissolved  in  brine  and  precipitated  by  dialysis,  gave  preparation  14,  weighing 
2.8  grams.  As  the  salt-saturated  solution  from  which  this  preparation  had 
separated  contained  so  little  protein,  it  appears  that  nearly  all  the  protein 
precipitated  from  the  aqueous  extract  by  saturating  with  sodium  chloride  had 
been  converted  into  the  insoluble  substance  forming  preparation  13. 

The  filtrate  from  the  salt-saturated  precipitate  produced  in  the  aqueous 
extract  was  dialyzed  in  water  for  several  days,  and  the  still  clear  solution 
then  dialyzed  in  alcohol  for  24  hours.  The  protein  thereby  precipitated  in 
a  coagulated  state  yielded  12.4  grams  of  preparation  15. 

Another  aqueous  extract  was  saturated  with  sodium  chloride,  and  the 
precipitate,  treated  in  the  same  way  as  preparation  13,  yielded  18  grams 
of  preparation  16. 

The  saline  washings  of  the  last  preparation  were  dialyzed  free  from  chlo- 
rides and  gave  a  precipitate  weighing  2.86  grams,  which  formed  preparation 
17,  having  the  properties  of  a  globulin,  dissolving  readily  on  adding  sodium 
chloride,  and  being  precipitated  from  such  solution  by  water. 

The  filtrate  from  the  final  precipitation  of  17,  when  heated  in  a  boiling 
water-bath,  gave  a  coagulum,  which  formed  preparation  18,  weighing  1.64 
grams. 

The  salt-saturated  filtrate  from  the  first  precipitation  of  16,  as  already 
described,  was  heated  to  boiling,  and  the  coagulum  produced  was  filtered  out, 
giving  preparation  19,  weighing  5.47  grams. 

Since  analysis  showed  that  most  of  the  preparations  already  described 
contained  phosphorus,  some  even  in  large  amount,  we  made  an  attempt  to 


26  THE    PROTEINS    OF   THE    WHEAT    KERNEL. 

separate  the  phosphorus  from  our  extract  in  order  to  determine,  if  possible, 
the  relation  of  the  preparations  free  from  phosphorus  to  those  which  con- 
tained much  phosphorus. 

2000  grams  of  meal  were  treated  with  6  liters  of  distilled  water,  and  the 
extract  (4  liters)  was  squeezed  out  as  completely  as  possible  in  a  press. 

As  a  preliminary  experiment  100  cc.  of  this  clear,  filtered  extract  were 
made  faintly  alkaline  to  phenolphthalein,  with  about  40  cc.  of  decinormal 
potassium-hydroxide  solution.  To  insure  a  sufficient  quantity  20  cc.  more 
of  alkali  were  added,  and  thereupon  a  little  calcium  chloride,  which  gave  a 
precipitate  that  seemed  to  partly  dissolve  on  adding  sodium  chloride.  The 
undissolved  part,  when  washed  with  dilute  sodium-chloride  solution,  water, 
and  alcohol,  and  dried,  formed  1.7  per  cent  of  the  meal,  contained  about  55 
per  cent  of  organic  matter,  and  left  45  per  cent  of  ash,  consisting  of  tricalcium 
phosphate. 

To  2000  cc.  of  the  original  extract  were  then  added  1350  cc.  of  a  solution 
containing  alkali,  equivalent  to  1560  cc.  decinormal  solution,  with  sodium 
chloride  enough  to  form  6.5  per  cent  of  the  total  liquid.  To  this  a  solution 
of  calcium  chloride  was  added  as  long  as  a  precipitate  formed,  and  after 
standing  over  night  the  solution  was  decanted  from  the  precipitate  and  filtered 
clear  on  a  pulp  filter.  Of  the  clear  filtrate  2200  cc.  were  made  as  neutral  as 
possible  to  litmus  by  adding  180  cc.  of  decinormal  hydrochloric  acid  solution. 
Of  the  solution  thus  neutralized  1000  cc.,  when  gradually  heated  in  a 
water-bath,  became  turbid  at  52°  and  a  considerable  coagulum  separated 
at  53°. 

After  the  temperature  had  been  slowly  raised  to  65°  and  kept  at  this  point 
some  time,  the  coagulum  was  filtered  out,  washed,  and  dried  as  usual,  giving 
preparation  20,  weighing  6.4  grams.  Another  portion  of  this  extract,  filtered 
from  the  calcium-chloride  precipitate,  was  saturated  with  ammonium  sulphate 
while  still  slightly  alkaline  to  litmus,  the  resulting  precipitate  filtered  out, 
dissolved  in  water,  its  solution  filtered  clear,  and  dialyzed.  A  slight  precip- 
itate formed  on  dialysis,  which  was  removed  by  filtering  and  the  solution 
heated  in  a  boiling  water-bath.  The  protein  thus  coagulated  weighed  3.07 
grams,  preparation  21. 

To  determine  what  effect  the  removal  of  the  phosphorized  substance  thrown 
out  by  calcium  chloride  had  upon  the  precipitation  with  sodium  chloride,  we 
made  neutral  to  litmus  a  liter  of  the  filtrate  from  the  calcium-chloride  precip- 
itate and  then  saturated  it  with  sodium  chloride.  The  large  precipitate 
which  formed  was  washed  by  decantation  with  water,  in  which  it  gradually 
dissolved,  until  only  an  insignificant  quantity  remained.  The  similarly  ob- 
tained precipitate  from  the  simple  aqueous  extract  we  have  shown  to  be  nearly 
all  insoluble  in  water. 


EXPERIMENTAL. 


To  separate  globulin  from  the  aqueous  extract  1200  cc.  of  clear,  filtered 
extract  were  obtained  from  200  grams  of  the  germ  meal  treated  with  2000  cc. 
of  water.  1000  cc.  of  this  extract  were  dialyzed  in  running  water  for  6 
days,  and  the  large  precipitate  resulting  gave  preparation  22,  weighing  9.17 
grams. 

These  preparations,  thus  variously  obtained  from  the  aqueous  extract, 
were  dried  to  constant  weight  at  110°  and  analyzed  with  the  results  given 
in  table  2,  most  of  the  figures  being  the  average  of  closely  agreeing  duplicate 
determinations. 


2.  —  Composition  of  preparations  of  protein  Jrom  the  water  extract  of 
the  wheat  embryo. 


6. 

7- 

8. 

9- 

10. 

II. 

12. 

13- 

14. 

Carbon  

P.  ct. 

SI.  13 

P.ct. 
CO  S2 

P.ct. 
SO  17 

P.ct. 

S2.3q 

P.ct. 

SI.  77 

P.ct. 

S2.I3 

P.ct. 

S2.73 

P.ct. 
4.3.  SQ 

P.ct. 
S2.28 

Hydrogen  .  .  . 
Nitrogen  
Sulphur  

6.85 
16.28 

1.18 

6.81 
16.47 
1.17 

7.01 
16.66 
I.OO 

6.83 
1  6.  20 

1.32 

6.81 
16.11 
i.  ^o 

7.04 
16.48 

I.4Q 

7.11 
1  6.  oo 
I.  S3 

5-77 
15-16 
o.qo 

6.97 
16.38 
1.39 

Phosphorus  .  . 
Ash  

0.72 

2.73 

0.97 

2.QO 

0.91 

3.  03 

trace 
o.3S 

0.17 
I.-zq 

0.06 
0.43 

none 

O.3q 

3-38 

I-1.O4. 

0.07 
0.44 

P2O5  in  ash  .  . 

1.88 

2.09 

1.91 

trace 

0.47 

trace 

none 

6-73 

trace 

15- 

16. 

17- 

18. 

19- 

20. 

21. 

22. 

Carbon.  ...... 

P.ct. 
SI   21 

P.ct. 
46  67 

P.  ct. 

si  87 

P.ct. 

P.ct. 
Si.qi 

P.ct. 
si  6s 

P.  Ct. 
S2.O2 

P.  ct. 
4Q.  SQ 

Hydrogen  .  .  . 

6.8s 

6.  IQ 

6.80 

6.86 

6.66 

7.OO 

6.68 

Nitrogen  

16.18 

IS.  80 

1  6.  6s 

16.  31 

16.08 

16.02 

I6.4S 

16.34 

Sulphur  

I.IO 

O.Q1* 

I.IQ 

I.3S 

i.  60 

1.  13 

1.24 

O.QI 

Phosphorus  .  . 

0.46 

2.S* 

trace 

trace 

trace 

trace 

none 

i.  8s 

Ash  

2   IQ 

8  17 

o  1.8 

o  4S 

0.32 

i  oq 

o.  s6 

2.  SO 

PjO5  in  ash.  . 

I.  II 

S-7I 

trace 

trace 

trace 

trace 

none 

1.  79 

Assuming  that  those  of  the  foregoing  preparations  which  contain  phos- 
phorus are  compounds  of  protein  with  the  nucleic  acid  which  has  been 
separated  from  the  aqueous  extract  of  wheat  germs,1  and  also  assuming  that 
all  the  phosphorus  of  these  preparations  is  a  part  of  the  nucleic  acid,  the 
composition  of  these  preparations  was  calculated  free  from  nucleic  acid. 
The  analyses  were  further  calculated  ash-free  by  subtracting  the  P2O5 
contained  in  the  ash  from  the  total  ash,  which  seems  permissible,  since 
the  ash  consisted  almost  wholly  of  metaphosphates  of  potassium  and  sodium, 

1  Osborne  &  Campbell,  Journal  American  Chemical  Society,  1900,  xxn,  p.  379 ;  also 
Osborne  &  Harris,  Report  Connecticut  Agricultural  Experiment  Station  for  1901,  p.  365. 


28 


THE    PROTEINS   OF   THE    WHEAT    KERNEL. 


strongly  indicating  that  the  P2O5was  derived  from  the  nucleic  acid.     These 
calculations  gave  the  results  shown  in  table  3. 

TABI,E  3. — Composition  of  leucosin  contained  in  the  preparations  from  water 
extracts  of  the  wheat  embryo. 


6. 

7- 

8. 

9- 

10. 

ii. 

12. 

13- 

14. 

Carbon  

P.ct. 
52.cn 

P.ct. 

'52.7'; 

P.ct. 
52.41 

P.ct. 
52.57 

P.ct. 
52  57 

P.ct. 

52.4.7 

P.ct. 
52.  Ql 

P.ct. 
51.21 

P.ct. 
52  64 

Hydrogen  .  .  . 
Nitrogen  
Sulphur  

7.12 
16.45 
1.  20 

7.16 

16.68 

1.12 

7.38 
16.94 

I.I* 

6.85 
16.26 
1.12 

6.91 
16.27 
1.14 

7.08 
16.55 

1.  50 

7.13 

16  06 
1.51 

7.09 
16.30 
1.  60 

7.02 
16.46 

1.  41 

Oxygen.  .  , 

22.21 

22.09 

22.14 

21.OO 

22.  QI 

22.40 

22.15 

21.78 

22.47 

IOO.OO 

IOO.OO 

100.00 

IOO.OO 

IOO.OO 

IOO.OO 

100.00 

100.00 

IOO.OO 

15. 

16. 

17- 

18. 

19- 

20. 

21. 

22. 

Carbon  

P.ct. 

52.61 

P.ct. 
52.44 

P.ct. 
52.06 

P.ct. 

P.ct. 
52.11 

P.ct. 
52.16 

P.ct. 
52.10 

P.ct. 
51.45 

Hydrogen  .  .  .  . 

7.06 

7.  IO 

6.Q2 

6.88 

6.71 

7.04 

7.1O 

Nitrogen 

l6.AO 

16  26 

16.71 

16.18 

16.13 

1  6.  20 

16  54 

',3 

16.57 

Sulphur  

1.17 

1.14 

I.IQ 

1.^5 

1.  60 

1.  14 

1.24 

I.l6 

Oxveren  .  . 

22.74 

22.86 

21.12 

21.28 

21.77 

22.88 

21.52 

IOO.OO 

IOO.OO 

IOO.OO 

IOO.OO 

IOO.OO 

IOO.OO 

IOO.OO 

Of  these  preparations,  6,  7,  8,  9,  18,  19,  20,  and  21  were  obtained  by  coagu- 
lation with  heat,  10  and  15  by  coagulation  with  alcohol,  13  and  16  by  satu- 
ration with  sodium  chloride,  n,  12,  14,  and  17  by  dialyzing  salt  solutions 
in  water,  and  22  by  direct  dialysis  of  the  aqueous  extract.  Since  some  of 
these  preparations  formed  a  large  part  of  the  protein  contained  in  the  extract, 
while  others  represented  fractions  of  it,  it  is  evident  that  all  contain  pro- 
tein of  the  same  composition,  mostly  combined  with  various  proportions  of 
nucleic  acid. 

Eliminating  the  nucleic  acid,  it  thus  appears  that  the  composition  of  the 
protein  part  of  those  preparations  which  contain  phosphorus  is  the  same  as 
that  of  the  phosphorus-free  protein  preparations,  although  the  former  con- 
tain from  very  little  up  to  more  than  37  per  cent  of  nucleic  acid. 

Most  of  these  preparations  might,  in  accordance  with  custom,  be  called 
nucleoproteids,  while  13  and  i6are,  both  in  properties  and  composition,  very 
much  like  nuclein.  It  is  probable  that  these  nucleoproteids  and  nucleins  are 
nucleic  acid  compounds  of  one  and  the  same  protein. 

It  is  to  be  noted  that  these  preparations  show  very  diverse  properties. 
Some  are  like  albumin;  some  like  globulin;  some  are  precipitated  by  satura- 


EXPERIMENTAL.  29 

tion  with  salt,  while  others  are  not.  As  we  have  shown,  these  different  prop- 
erties are  the  result  of  changes  caused  by  varying  the  conditions  under  which 
the  protein  exists  in  the  extract,  and  depend  chiefly  on  the  degree  of  acidity 
of  the  extract,  whereby  the  numbers  and  kinds  of  acid  molecules  that  com- 
bine with  the  protein  molecule  are  altered.  „ 

Whatever  may  be  the  true  cause  of  these  changes,  it  is  evident  from  the  / 

results  here  described  that  the  distinctions  heretofore  made  between  globulin 
and  albumin,  myosin,  and  vitellin,  etc.,  have  very  little  value  as  a  basis 
for  classifying  protein  substances.  This  explains  the  difference  between 
O'Brien's  classification  of  leucosin  as  a  myosin-like  globulin,  to  which  refer- 
ence was  made  at  the  beginning  of  this  paper,  and  our  designation  of  it  as 
an  albumin,  because  of  the  ready  solubility  in  water  and  coagulability  by  heat 
of  the  preparations  which  we  had  made. 

Thus  preparation  22,  weighing  9.17  grams,  was  insoluble  in  water  and  in 
salt  solution,  and  was  not  a  precipitate  of  globulin,  since  in  the  filtrate  from 
which  it  had  separated  on  dialysis  only  0.87  gram  of  coagulable  albumin 
was  found  instead  of  9.5  grams,  as  usually  found  by  direct  coagulation  of 
the  aqueous  extracts  ;  moreover,  the  analysis  indicates  that  it  is  a  compound 
of  leucosin,  with  20  per  cent  of  nucleic  acid. 

On  the  preceding  pages  it  was  shown  that  a  small  part  of  the  precipitate, 
produced  by  saturating  the  aqueous  extract  with  sodium  chloride,  is  soluble 
in  dilute  salt  solution,  and  can  be  precipitated  from  this  solution  by  dialysis, 
as  a  globulin-like  substance,  readily  soluble  again  in  salt  solution.  This 
globulin-like  substance  contains  little  or  no  nucleic  acid,  and  has  very  nearly 
the  same  elementary  composition  as  leucosin,  of  which  it  is  probably  a  com- 
pound with  a  small  proportion  of  some  body  of  low  molecular  weight. 

It  is  plain  from  these  facts  that  O'Brien's  myosin  contains  the  same  pro- 
tein substance  as  my  leucosin. 

O'Brien's  "albumin,"  coagulating  at  75°  to  80°,  is  probably  more  of  this 
same  leucosin,  as  shown  by  preparation  8,  which  formed  about  25  per  cent 
of  the  total  coagulable  protein.  The  experience  of  the  writer  has  been  that 
complete  coagulation,  especially  in  a  solution  nearly  free  from  salts,  can  be 
effected,  if  at  all,  only  by  heating  the  solution  much  above  the  lower  coagu- 
lation temperature  of  the  protein  to  be  separated. 

From  the  whole  seed  leucosin  was  obtained  with  the  same  composition  and 
general  properties  as  from  the  embryo,  but  preparations  from  the  whole  seed 
were  free  from  phosphorus.  This  was  probably  because  the  proportion  of 
nucleic  acid  to  protein  matter  was  smaller  in  the  whole  seed  than  in  the  embryo, 
so  that  on  extracting  with  water  the  nucleic  acid  did  not  form  soluble  com- 
pounds with  the  leucosin,  but  remained  undissolved  in  combination  with  the 
other  proteins.  In  table  4,  on  the  following  page,  is  given  the  average  of 
analyses  of  albumin  from  the  cereals. 


THE    PROTEINS   OF   THE    WHEAT    KERNEL. 
TABLE  4. — Composition  of  albumin  prepared  from  various  cereals. 


Wheat 
embryo. 

Wheat 
kernel. 

Rye 

kernel. 

Barley 
kernel. 

Barley 
malt. 

Carbon  

P.ct. 
52.6s 

P.ct. 
5^.02 

P.ct. 

S2.Q7 

P.ct. 

S2.8i 

P.ct. 
c-j.o7 

Hydrogen  

7.04. 

6.84 

6.7Q 

6.78 

6.72 

Nitrogen  

i6.4^ 

16.80 

1  6.  66 

1662 

I6.7I 

Sulphur  

1.^2 

1.28 

I.^S 

1.47 

1 

Oxveren  .  . 

22.  S6 

22.06 

22  21, 

22.^2 

y  23.50 

IOO.OO 

IOO.OO 

IOO.OO 

100.00 

IOO.OO 

In  an  earlier  paper  on  the  "Chemical  Nature  of  Diastase "  *  I  pointed  out 
that  diastatic  action  appeared  to  be  always  associated  with  leucosin.  Since 
extracts  of  wheat  embryo  were  so  rich  in  leucosin,  the  diastatic  power  of  the 
germ  meal  was  determined  by  extracting  with  four  times  its  weight  of  water, 
and,  under  the  conditions  of  L,itner's  test,  o.  10  cc.  of  the  extract  so  made, 
when  added  to  10  cc.  of  a  2  per  cent  solution  of  soluble  starch,  formed  within 
i  hour,  at  20°,  enough  sugar  to  reduce  5  cc.  of  Fehling's  solution.  The 
o.io  cc.  of  extract  corresponds  to  25  mg.  of  the  germs,  from  which  it  is  seen 
that  this  meal  possesses  high  diastatic  power,  though  it  is  inferior  in  this 
respect  to  active  malt. 

HYDROLYSIS    OF  LEUCOSIN. 

In  order  to  determine  the  proportion  of  the  different  products  which  are 
formed  by  boiling  proteins  with  strong  acids,  it  is  necessary  to  use  relatively 
large  amounts  of  the  protein.  Commercial  wheat-germ  meal  was  therefore 
used  in  order  to  obtain  a  sufficient  quantity  of  leucosin  for  this  purpose. 
The  freshly  ground  meal  was  extracted  with  water,  and,  as  the  gummy 
solution  could  not  be  filtered  within  a  reasonable  time,  an  equal  volume  of 
saturated  ammonium  sulphate  solution  was  added.  The  precipitate  thus 
produced  was  filtered  out,  dissolved  in  water,  the  solution  filtered  perfectly 
clear,  and  the  leucosin  coagulated  by  heating  the  dilute  solution  to  65°  in  a 
water-bath  at  70°.  As  the  only  other  protein  substance  present  in  this  solu- 
tion was  a  relatively  insignificant  quantity  of  proteose,  the  product  obtained 
was  practically  free  from  any  other  protein.  This  coagulum  was  thoroughly 
washed  with  hot  water,  in  order  to  remove  any  admixed  proteose,  and  dehy- 
drated with  absolute  alcohol.  The  preparation  formed  a  light  white  powder. 

Owing  to  the  difficulty  of  preparing  large  quantities  of  this  protein,  which 
occurs  in  very  small  quantity  in  the  wheat  kernel,  we  were  limited  in  this 


1  Osborne,  Journal  American  Chemical  Society,  1895,  x-vil,  p.  587. 


EXPERIMENTAL. 


hydrolysis  to  257.8  grams  of  water  and  ash-free  leucosin.  This  was  hydro- 
lyzed  and  the  glutaminic  acid  separated  as  hydrochloride.  When  dried  in 
vacuo  over  sulphuric  acid,  12.39  grams  of  the  hydrochloride  were  obtained, 
which  melted  at  198°  with  effervescence. 

Nitrogen  :  0.5482  gram  substance  gave  NH3  =  4.i7  cc.  HC1  (i  cc.  HC1  =  o.oi  gram  N). 

Chlorine :  0.2528  gram  substance  gave  0.1989  gram  AgCl. 

Calculated  for  C5H10O4NC1,  N  7.64,  Cl  19.35  p.  ct;  found,  N  7.61,  Cl  19.45  p.  ct. 

The  filtrate  from  the  glutaminic  acid  hydrochloride  was  concentrated  to  a 
sirup  under  reduced  pressure,  the  residue  taken  up  in  alcohol  and  saturated 
with  dry  hydrochloric  acid  gas.  The  solution  was  then  evaporated  to  a 
thick  sirup  under  reduced  pressure,  the  residue  again  esterified  with  alcohol 
and  hydrochloric  acid,  and  the  solution  concentrated  as  before.  The  esteri- 
fication  was  again  repeated,  the  final  concentration  being  made  at  a  pressure 
of  10  mm.  from  a  bath,  the  temperature  of  which  did  not  rise  above  40°. 
The  free  esters  of  the  amino-acids  were  then  liberated  from  the  residue, 
extracted  with  ether,  and  dried  with  potassium  carbonate  and  anhydrous 
sodium  sulphate  in  the  usual  way.  The  aqueous  layer  was  then  made 
strongly  acid  with  hydrochloric  acid  and  the  salts  removed  by  concentra- 
tion and  treatment  with  alcoholic  hydrochloric  acid.  The  alcoholic  extracts 
containing  the  hydrochlorides  of  the  amino-acids  were  evaporated  to  a  thick 
sirup  under  reduced  pressure  and  the  residue  esterified  as  above  described. 
The  free  esters  were  then  liberated  and  their  ether  solution  dried  as  before. 
After  distilling  off  the  ether  on  the  water-bath,  at  atmospheric  pressure,  the 
residue  was  distilled  with  the  following  results  : 


Temperature 

Fraction. 

of  bath 

Pressure. 

Weight. 

up  to  — 

o 

mm. 

Grams. 

I 

75 

12.0 

21.76 

II 

80 

IO.O 

25.86 

III 

125 

0.8 

49.60 

IV 

200 

0.8 

49-39 

146.61 

'•} 


f  Fraction  I.     Temperature  of  bath  up  to  75 
|  Pressure,  12  mm.     Weight,  21.76  grams. 

This  fraction  was  saponified  directly  after  collection  by  evaporating  on  the 
water- bath  with  concentrated  hydrochloric  acid.  The  residue  was  esterified 
with  alcohol  and  hydrochloric  acid  and  allowed  to  stand  for  several  days 
on  ice.  The  glycocoll  ester  hydrochloride  which  separated  weighed  1.73 
grams  and  melted  at  145°. 


32  THE    PROTEINS   OF   THE    WHEAT    KERNEL. 

Nitrogen:  0.2851  gram  substance,  dried  in  vacuo,  gave   NH3  =  2.88  cc.  HC1    (i    cc. 

HCl  =  o.oi  gram  N). 

Chlorine:  0.2843  gram  substance,  dried  in  vacuo,  gave  0.2889  gram  AgCl. 
Calculated  for  C4H10OS  NCI,  N  10.05,  Cl  25.40  p.  ct.;  found,  N  10.12,  Cl  25.12  p.  ct. 

In  the  filtrate  from  the  glycocoll  ester  hydrochloride  the  free  amino-acids 
were  regenerated  and  subjected  to  fractional  crystallization  in  water  and 
alcohol.  There  were  obtained  3.06  grams  of  nearly  pure  alanine. 

Carbon  and  hydrogen:  0.3682  gram  substance  gave  0.5460  gram  COa   and  0.2649 

gram  H2O. 

Nitrogen  :  0.2508  gram  substance  gave  NHS  =  3.92  cc.  HC1  (i  cc.  HC1  =  o.oi  gram  N). 
Calculated  for  C3H7O,N,  C  40.40,  H  7.93,   N  15.75  P-  ct.;  found,  C  40.44,  H  7.99, 

N  15-63  p.  ct. 

The  alanine  decomposed  at  290°. 

|  Fraction  II.     Temperature  of  bath  up  to  80°.  ) 
{  Pressure,  10  mm.     Weight,  25.86  grams.          j 

After  saponifying  with  boiling  water,  the  solution  of  the  amino-acids  was 
evaporated  to  dryness  under  reduced  pressure.  The  dried  residue,  which 
weighed  18.78  grams,  was  extracted  with  boiling  alcohol,  whereby  1.68 
grams  went  into  solution.  From  the  part  insoluble  in  alcohol  there  were 
isolated,  by  fractional  crystallization,  5.53  grams  of  leucine,  5.79  grams 
alanine,  and  0.47  gram  of  substance  which  had  the  percentage  composition 
of  amino-valerianic  acid. 

Carbon  and  hydrogen:   0.2094  gram  substance  gave  0.3913  gram   CO,  and  0.1793 

gram  H2O. 
Calculated  for  C5HUOUN,  C  51.22,  H  9.48  p.  ct.;  found,  C  50.96,  H  9.51  p.  ct. 

From  the  more  soluble  part  of  this  fraction  glycocoll  was  isolated  as  the 
hydrochloride  of  the  ester.  This  weighed  2.78  grams,  equivalent  to  1.50 
grams  of  glycocoll,  and  melted  at  144°  to  145°. 

f  Fraction  III.     Temperature  of  bath  up  to  125°.  \ 
(  Pressure,  0.8  mm.     Weight,  49.6  grams.  j 

Fraction  III  was  saponified  by  boiling  with  10  parts  of  water  for  10  hours, 
and  the  solution  evaporated  to  dryness  under  reduced  pressure.  After 
extracting  the  proline  with  boiling  alcohol  the  insoluble  part  was  fractionally 
crystallized.  There  were  obtained  23.72  grams  of  leucine  and  2.62  grams 
of  alanine.  The  isolated  leucine  decomposed  at  298°. 

Carbon  and  hydrogen:  0.1835  gram  substance  dried  at  110°  gave  0.3670  gram  CO2  and 

0.1670  gram  H2O. 

Nitrogen:  0.3086  gram  substance  gave  NHS  =  3.34  cc.  HC1  (i  cc.  HCl  =  o.oi  gram  N). 
Calculated  for  C6H13O2N,  C  54.89,  H  10.01,  N  10.70  p.  ct;  found  C  54.63,  H  10.11, 

N  10.82  p.  ct. 


EXPERIMENTAL.  33 

The  combined  alcoholic  solutions  from  fractions  II  and  III,  which  con- 
tained the  proline,  were  evaporated  to  dry  ness  under  reduced  pressure,  and 
the  residue  extracted  with  boiling  alcohol,  in  which  2.3  grams  did  not  dis- 
solve. The  solution  filtered  from  this  was  again  evaporated  to  dryness 
under  reduced  pressure,  the  residue  dissolved  in  water,  and  the  copper  salts 
prepared  by  boiling  for  an  hour  with  an  excess  of  copper  hydroxide.  The 
deep  blue  solution  was  evaporated  to  dryness  under  reduced  pressure,  and 
the  residue  boiled  with  absolute  alcohol,  which  dissolved  the  /-proline  copper 
salt.  The  residue  of  racemic  copper  salt,  insoluble  in  alcohol,  was  dissolved 
in  water  and  the  solution  concentrated.  Of  racemic  proline  copper  2.41 
grams  were  obtained,  equivalent  to  1.69  grams  of  «-proline. 

Water:  0.3190  gram  substance,  air-dried,  lost  0.0353  gram  H2O  at  110°. 
Copper:  0.2827  gram  substance,  dried  at  110°,  gave  0.0766  gram  CuO. 
Calculated  for  C10H16O4N3Cu.  2  H2O,  H2O  11.00  p.  ct.  ;  found,  H2O  11.07  P-  ct. 
Calculated  for  C,0H16O4N2Cu,  Cu  21.79  P-  ct-  >  found,  Cu  21.65  P-  ct- 

The  alcoholic  solution  of  the  copper  salt  of  the  /-proline  was  evaporated 
to  dryness  under  reduced  pressure.  The  dried  residue  weighed  8.24  grams, 
equivalent  to  6.5  grams  of  /-proline. 

For  identification  a  small  portion  was  freed  from  copper  with  hydrogen 
sulphide,  and  the  free  proline  converted  into  the  phenylhydantoin,  accord- 
ing to  the  directions  of  Hmil  Fischer.1 

The  hydantoin  melted  sharply  at  143°  and  gave  the  following  analysis  : 

Carbon  and  hydrogen :  0.2334  gm.  substance  gave  0.5676  gm.  CO2  and  0.1204  gm.  H2O. 
Nitrogen  :  0.1373  gram  substance  gave  NH3  =  1.78  cc.  HC1  (i  cc.  HC1  =  o.oi  gram  N). 
Calculated  for  C12HnO2N2,  C  66.60,  H  5.61,  N  12.99  p.  ct.  ;  found,  C  66.32,  H  5.73,  N 
12.96  p.  ct. 

f  Fraction  IV.     Temperature  of  bath  up  to  200°.  ) 
\  Pressure,  0.8  mm.     Weight,  49.39  grams.  j 

From  fraction  IV  the  ester  of  phenylalanine  was  removed  in  the  usual 
manner  by  shaking  out  with  ether,  and  after  freeing  from  ether  the  residual 
ester  was  saponified  by  dissolving  in  concentrated  hydrochloric  acid  and 
evaporating  on  the  water-bath.  Of  phenylalanine  hydrochloride  12.12 
grams  were  obtained,  which  is  equivalent  to  9.93  grams  of  phenylalanine. 
For  identification  the  hydrochloride  was  recrystallized  from  strong  hydro- 
chloric acid  and  converted  into  the  free  acid  by  evaporation  with  excess  of 
ammonia.  When  once  recrystallized  from  water,  it  melted  at  263°  to  265°. 

Carbon  and  hydrogen :  0.2962  gram  substance,  dried  at  110°,  gave  0.7089  gram  CO2  and 

0.1801  gram  H2O. 

Nitrogen :  o.  1924  gram  substance  gave  NH3  =  1.65  cc.  HC1  ( i  cc.  HC1  =  o.oi  gram  N). 
Calculated  for  C9HUO2N,  C  65.39,  H  6-73»  N  8-5°  P-  ct-  5  found,  C  65.27,  H  6.76,  N 

8.57  P-  ct. 

Fischer,  E.,  Zeitschrift  fur  physiologische  Chemie,  1901,  xxxm,  p.  251. 
3 


34  THS    PROTEINS    OF   THE    WHEAT    KERNEL. 

The  aqueous  layer  was  saponified  by  warming  with  an  excess  of  barium 
hydroxide  on  the  water-bath  for  7  hours.  After  standing  for  some  time 
the  crystals  of  racemic  barium  aspartate  were  filtered  out  and  decomposed 
with  an  equivalent  quantity  of  sulphuric  acid.  The  filtrate  from  the  barium 
sulphate  gave,  on  concentration,  5.06  grams  of  aspartic  acid,  which,  when 
recrystallized  from  water,  was  analyzed. 

Carbon  and  hydrogen:  0.2483  gram  substance,  dried  at  110°,  gave  0.3278  gram  CO.,  and 

0.1215  gram  H2O. 

Nitrogen  :  0.2769  gram  substance  gave  NH3  =  2.93  cc.  HC1  (i  cc.  HC1  =  o.oi  gram  N). 
Calculated  for  C4HTO4N,  C  36.06,  H  5.31,  N  10.55  p.  ct.  ;  found,  C  36.00,  H  5.44,  N 

10.59  P-  ct. 

The  filtrate  from  barium  aspartate  was  freed  from  barium  quantitatively 
with  sulphuric  acid  and  the  filtrate  from  the  barium  sulphate  concentrated  to 
small  volume  and  saturated  with  hydrochloric  acid  gas.  After  long  standing 
on  ice,  2.15  grams  of  glutaminic  acid  hydrochloride  separated. 

The  filtrate  from  this  was  evaporated  under  reduced  pressure,  the  residue 
taken  up  in  water,  and  the  chlorine  removed  with  silver  sulphate. 

After  removing  the  sulphuric  acid  with  an  equivalent  quantity  of  barium 
hydroxide,  the  solution  was  boiled  with  an  excess  of  copper  hydroxide.  The 
filtered  solution,  on  standing,  separated  tyrosine-like  needles  of  copper  laevo- 
aspartate  which  weighed  7.41  grams,  equivalent  to  3.57  grams  of  aspartic 
acid. 

Nitrogen:  0.1853  gram  substance,  air-dried,  gave  NH3  =  0.93  cc.  HC1  (i  cc.  HCl  =  o.oi 

gram  N). 

Copper  :  0.1677  gram  substance,  air-dried,  gave  0.0491  gram  CuO. 
Calculated  for  C4H5O4N,  Cu  •  4^  H2O,  N  5.09,  Cu  23.06  p.  ct.;  found,  N5.O2,  Cu  23.37 

p.  ct. 

From  the  copper  salt  the  free  acid  was  regenerated  and  analyzed. 

Carbon  and  hydrogen:  0.2041  gram  substance  gave  0.2715  gram  CO3  and  0.1054  gram 

H2O. 
Calculated  for  C4H7O4N,  C  36.06,  H  5.31  p.  ct.;  found,  C  36.29,  H  5.74  p.  ct. 

Specific  rotation.  —  Dissolved  in  20  per  cent  hydrochloric  acid, 

(a)  ^  =  +23.8° 

Fischer  &  Dorpinghaus1  found 


An  effort  to  isolate  serine  in  the  filtrate  from  the  copper  aspartate  failed. 
Fischer  &  Dorpinghaus,  Zeitschrift  fur  pbysiologische  Chemie,  1902,  xxxvi,  p.  462. 


EXPERIMENTAL.  35 

THE  RESIDUE  AFTER  DISTILLATION. 

The  residue  remaining  after  distillation  of  the  esters  weighed  57  grams. 
This  was  dissolved  in  boiling  alcohol,  and  after  cooling  1.98  grams  of  needle 
crystals  were  filtered  out.  The  filtrate  was  evaporated  to  a  sirup  under 
reduced  pressure,  saponified  by  heating  with  an  excess  of  barium  hydroxide, 
and,  after  removing  the  barium,  evaporated  to  small  volume  under  reduced 
pressure.  The  solution  was  then  saturated  with  hydrochloric  acid,  and, 
after  standing  on  ice  for  a  long  time,  yielded  7.39  grams  of  glutaminic  acid 
hydrochloride.  The  free  acid  prepared  by  evaporating  with  an  exactly 
equivalent  quantity  of  potassium  hydroxide,  when  recrystallized  from  water, 
melted  at  202°  to  203°  with  effervescence. 

Carbon  and  hydrogen  :  0.3646  gram  substance,  dried  at  110°,  gave  0.5432  grain  CO3  and 
0.2019  gram  H2O. 

Nitrogen:  0.3696  gram  substance  gave  NH3=3.55cc.  HC1  (i  cc.  HCl  =  o.oi  gram  N). 

Calculated  for  C5H9O4N,  C  40.82,  H  6.12,  N  9.52  p.  ct.;  found,  C  40.63,  H  6.15,  N  9.60 
p.  ct. 

The  total  glutaminic  acid  obtained  from  leucosin  was  17.5  grams,  or  6.73 
per  cent.  This  result  is  higher  than  that  recently  recorded  in  this  labora- 
tory, namely,  5. 72.* 

This  protein  is  one  from  which  the  glutaminic  acid  hydrochloride  can  be 
directly  obtained  only  with  great  difficulty.  In  the  former  paper  attention 
was  directed  to  this  fact,  and  the  statement  made  that  it  is  possible  that  the 
result  given  was  too  low. 

TYROSINE. 

Forty  grams  of  leucosin,  equal  to  34.96  grams  dried  at  110°,  were  boiled 
for  12  hours  with  a  mixture  of  120  grams  of  sulphuric  acid  and  240  grams 
of  water.  After  removing  the  sulphuric  acid  with  an  equivalent  quantity  of 
barium  hydroxide,  the  solution  was  evaporated  with  an  excess  of  barium 
carbonate  in  order  to  remove  ammonia.  After  removing  the  barium,  the 
solution  was  concentrated  to  a  small  volume  on  the  water-bath  and  allowed 
to  stand  for  some  time.  The  substance  which  separated  was  washed  with 
cold  water,  dissolved  in  ammonia,  the  solution  treated  with  bone-black,  and 
evaporated.  On  cooling,  i  .0360  grams  tyrosine  separated  in  colorless  needles. 
The  filtration  from  this,  on  further  concentration,  yielded  0.13  gram  more 
tyrosine,  making  a  total  of  1. 1660  grams,  or  3.33  per  cent.  This  was  recrys- 
talized  and  analyzed. 

Carbon  and  hydrogen :  0.4573  gram  substance,  dried  at  110°,  gave  0.9994  gram  CO2  and 
0.2813  gram  H2O. 

Calculated  for  C9HUO3N,  C  59.62,  H  6.13  p.  ct.;  found,  C  59.60,  H  6.11  p.  ct. 

1  Osborne  &  Gilbert,  American  Journal  of  Physiology,  1906,  xv,  p.  333. 


36  THE;  PROTEINS  OF  THE;  WHEAT 

ARGININE,  HISTIDINE,  AND  L,YSINE. 

The  filtrate  from  the  tyrosine,  by  Kossel's  method,  yielded  a  solution  in 
which  the  nitrogen  found  corresponded  to  0.99  gram  of  histidine,  or  2.83 
per  cent. 

The  identity  of  the  histidine  could  not  be  established  owing  to  its  very 
small  amount. 

The  solution  of  the  arginine  contained  nitrogen  equal  to  0.6720  gram, 
which  is  equal  to  2.08  grams  of  arginine,  or  5.94  per  cent.  A  part  was  con- 
verted into  the  nitrate  and  this  latter  into  the  copper  salt  by  boiling  with  an 
excess  of  copper  hydroxide  and  the  copper  salt  recrystallized  from  water. 

Water :  0.1984  gram  substance,  air-dried,  lost  0.0191  gram  H2O  at  110°. 
Copper :  0.1766  gram  substance,  dried  at  110°,  gave  0.0262  gram  CuO. 
Calculated  for  C12H28O4N8Cu  (NO3)2  •  3  H2O,  H2O  9.15  p.  ct;  found,  H2O  9.62  p.  ct. 
Calculated  for  C12H28O4N8Cu  (NO8)2,  Cu  11.85  P-  ct.;  found,  Cu  11.83  P-  ct. 

The  filtrate  from  the  copper  salt  was  freed  from  copper  and  the  solution 
evaporated  with  an  excess  of  sulphuric  acid  under  reduced  pressure.  The 
sulphuric  acid  was  removed  with  an  excess  of  barium  hydroxide,  and  the 
barium  with  carbonic  acid.  The  filtrate  from  the  barium  carbonate  was  evap- 
orated to  dryness  and  the  arginine  converted  into  the  picrolonate,  according 
to  the  directions  of  Steudel.  This  melted  at  226°  to  227°;  Steudel  gives  225°. 

Nitrogen:  0.0516  gram  substance,  dried  at  110°,  gave  n.8  cc.  moist  N,  at  25°  and 

765  mm. 
Calculated  for  C6HUO2N4  •  Ci0H8O5N4,  N  25.62  p.  ct.;  found,  N  25.71  p.  ct. 

The  filtrate  from  the  silver  precipitate  which  contained  the  arginine  and 
histidine  was  freed  from  silver  and  barium,  and  the  lysine  precipitated  with 
phosphotungstic  acid  and  then  converted  into  the  picrate,  of  which  2.47 
grams,  equal  to  0.9616  gram  of  lysine,  or  2.75  per  cent,  was  obtained. 
This  was  recrystallized  from  water  and  analyzed. 

Nitrogen:  0.2288  gram  substance  gave  38.6  cc.  moist  N2  at  25.5°  and  759  mm. 
Calculated  for  C6HHO2N2  •  C6H3O7N3,  N  18.70  p.  ct.;  found,  N  18.77  P-  ct. 

The  results  of  this  hydrolysis  are  given  in  table  5. 


TABI,E  5. — Leucosin. 


P.ct. 

Glycocoll 0.94 

Alanine 4.45 

Aminovalerianic  acid 0.18 

Leucine U-34 

c-proline 3.18 

Phenylalanine 3.83 

Aspartic  acid 3.35 

Glutamiuic  acid 6.73 


f.ct. 


Tyrosine 3.34 

Lysine 2.75 

Histidine 2.83 

Arginine 5.94 

Ammonia 1.41 

Tryptophane present 


50.32 


EXPERIMENTAL.  37 

PROTEINS   OP   WHEAT   FLOUR  SOLUBLE   IN  SODIUM-CHLORIDE   SOLUTION. 
THE  GZ,OBUI,IN  OF  WHBA.T  FI.OUR. 

Beside  the  proteins  soluble  in  water,  10  per  cent  sodium-chloride  brine 
extracts  from  ground  wheat  kernels  a  globulin  which  is  present  in  the  seed 
in  small  quantity.  Ten  kilograms  of  ' '  straight  flour ' '  were  extracted  with 
34  liters  of  10  per  cent  sodium-chloride  solution  by  suspending  the  flour  in 
the  liquid,  stirring  frequently,  and  then  allowing  the  whole  to  stand  at 
rest  over  night.  The  extract,  separated  from  the  flour  and  filtered  as  clear 
as  possible,  had  a  very  slight  acid  reaction,  was  of  a  pink  color,  very  viscid 
consistence,  and  formed  about  one-half  of  the  solution  added  to  the  flour. 
This  was  saturated  with  ammonium  sulphate,  and  the  resulting  precipitate 
filtered  off  and  dissolved  as  far  as  possible  in  4  liters  of  10  per  cent  sodium- 
chloride  solution.  The  exceedingly  viscid  solution  was  filtered  with  diffi- 
culty, placed  in  a  dialyzer,  and  left  in  a  stream  of  running  water  until  the 
chlorides  were  removed.  As  the  salts  dialyzed  out  the  globulin  gradually 
separated  in  minute  particles,  the  larger  of  these  being  evidently  spheroidal 
in  form.  This  precipitate  weighed  5.8  grams.  This  protein,  dissolved  in 
a  10  per  cent  sodium-chloride  solution,  when  heated  slowly,  gave  a  very 
slight  turbidity  at  87 °,  which  increased  slightly  up  to  99°.  On  boiling, 
some  coagulum  developed,  and  on  adding  acid  to  the  solution  filtered  from 
this  coagulum  a  very  considerable  precipitate  formed. 

Dilution  of  the  solution  of  the  globulin  in  10  per  cent  sodium-chloride 
brine  precipitated  the  protein.  Saturation  with  sodium  chloride  gave  no 
precipitate.  Saturation  with  magnesium  sulphate,  and  also  with  ammonium 
sulphate,  completely  precipitated  the  globulin.  When  dried  at  110°,  this 
preparation,  23,  gave,  when  analyzed,  the  results  which  are  shown  in  the 
table  on  page  38. 

Another  preparation,  24,  was  made  in  the  same  way  as  the  preceding,  ex- 
cept that  the  precipitation  with  ammonium  sulphate  was  omitted,  the  filtered 
extract  being  placed  at  once  in  dialyzers.  L,ike  the  preceding  solution,  this 
was  at  first  very  viscid,  but  after  the  removal  of  the  chloride  the  viscid 
property  was  entirely  lost.  This  viscidity  can  hardly  be  due  to  the  globulin, 
for  solutions  of  the  precipitated  globulin  showed  no  trace  of  it.  The  aqueous 
extract  of  the  flour  had  no  such  property,  and  it  is  difficult  to  say  to  what 
this  was  due  unless  to  the  presence  of  carbohydrate.  After  complete 
removal  of  the  chlorides  the  solution  was  filtered  from  the  precipitate,  the 
latter  dissolved  in  10  per  cent  sodium-chloride  solution,  and  the  insoluble 
matter  filtered  off.  The  residue  so  removed  consisted  chiefly  of  an  insoluble 
form  of  the  globulin.  This  was  dissolved  in  o.  2  per  cent  potassium-hydroxide 
water,  the  solution  filtered  clear,  and  precipitated  by  neutralization  with 


THE;  PROTEINS  OE  THE  WHEAT 


0.2  per  cent  hydrochloric  acid.  The  precipitate  weighed  2  grams.  The 
solution  of  the  globulin  was  dialyzed  till  free  from  chlorides  and  the  sepa- 
rated globulin  found  to  weigh  5  grams.  The  yield  in  this  extraction  was 
therefore  7  grams,  or  nearly  the  same  as  in  the  preceding.  The  composi- 
tion of  preparation  24  is  given  by  the  analysis  shown  in  the  following  table  : 

Preparations  23  and  24. 


Preparation  23. 

Preparation  24. 

I. 

II. 

Arerage. 

Ash- 
free. 

I. 

II. 

Average. 

Ash- 
free. 

Carbon  

P.ct. 

50.87 
6.74 
18.13 
0.97 

P.ct, 

50.79 
6.67 
18.25 

P.ct. 
50.83 
6.71 
18.19 
0-97 

P.ct. 
5L07 
6-75 
18.27 

0-97 

22.  QA 

P.  ct. 
50.77 
7-03 
18.43 
0.71 

P.ct. 

50.63 
6.84 
18.32 

P.ct. 
50.70 

6-93 
18.38 
0.71 

P.ct. 
51  01 

6-97 
18.48 
0.71 
22.83 

Hydrogen  
Nitrogen  

Sulphur  
Oxvsren  .  .  , 

Ash  

0.48 

0.62 





IOO.OO 

IOO.OO 

The  globulin  contained  in  the  "  shorts  "  from  the  spring  wheat  was  next 
extracted  by  treating  2000  grams  with  10  per  cent  sodium-chloride  solution 
for  3  hours,  with  frequent  stirring,  and  then  squeezing  out  the  extract 
in  a  screw-press.  After  the  suspended  starch  had  settled,  the  extract  was 
decanted  and  saturated  with  ammonium  sulphate.  The  precipitate  was  dis- 
solved in  10  per  cent  sodium-chloride  brine,  and  the  solution  filtered  from 
the  insoluble  matter.  The  clear  extract  was  then  dialyzed  till  free  from 
chlorides,  and  the  precipitated  globulin  found  to  consist  of  well-formed 
spheroids  and  masses  of  confluent  spheroids.  The  globulin  was  strongly 
colored,  since  much  coloring  matter  was  extracted  from  the  shorts,  render- 
ing the  extract  a  deep  red-brown  in  color.  The  precipitate  was  then  filtered 
off,  again  dissolved  in  the  salt  solution  and  placed  in  a  dialyzer.  When  free 
from  chlorides,  the  precipitated  globulin  was  filtered  off  and  found  to  be  still 
much  colored.  After  partly  washing  with  water  it  began  to  dissolve,1  and 
was  therefore  next  washed  with  dilute  alcohol  and  finally  with  absolute 
alcohol  and  with  ether.  Dried  over  sulphuric  acid,  it  weighed  2.22  grams, 
and  had  the  composition  shown  under  the  heading  ' '  Preparation  25  "  in  the 
table  on  page  39. 

1  This  solubility  of  a  globulin  is  due  to  the  formation  of  acid  compounds  which  are 
soluble  in  water,  but  insoluble  in  a  dilute  saline  solution.  Cf.  Osborne,  Journal  Amer- 
ican Chemical  Society,  1902,  xxiv,  p.  39. 


EXPERIMENTAL. 


39 


The  filtrate  and  washings  obtained  from  this  preparation  after  the  second 
precipitation  by  dialysis  were  precipitated  by  adding  a  few  drops  of  sodium- 
chloride  solution.  The  precipitate  produced,  which  was  nearly  free  from 
coloring  matter,  weighed  1.25  gram.  When  analyzed  it  gave  the  results 
shown  under  the  heading  "  Preparation  26"  in  the  accompanying  table. 

Preparations  25,  26,  and  27. 


Preparation  25. 

Preparation  26. 

Preparation  27. 

I. 

Ash- 
free. 

I. 

Ash- 
free. 

I. 

II. 

Average. 

Carbon 

P.ct. 

50-79 
6.80 
18.19 
0.66 

P.ct. 
51.00 
6.83 
18.26 
0.66 

2\.2^ 

P.ct. 

P.ct. 

P.ct. 

P.ct. 

P.ct. 

Hydrogen  .  .  . 
Nitrogen  .... 

18.56 

18.64 

18.15 

18.43 

18.29 

Ash 

O.47 

The  solution  filtered  from  this  preparation  was  next  heated  to  boiling 
and  the  coagulum  obtained,  which  weighed  i  gram,  when  analyzed  was  found 
to  contain  the  same  amount  of  nitrogen  as  the  globulin.  It  was  considered 
to  be  a  coagulated  globulin  which  had  been  held  in  solution  in  water  by 
acid.  Its  nitrogen  content  is  shown  under  the  heading  "  Preparation  27." 

The  total  quantity  of  globulin  obtained  from  the  shorts  was  in  this  case 
4.47  grams,  being  nearly  twice  as  much  as  was  similarly  obtained  from  a 
like  quantity  of  the  flour.1 

TABI,E  6. — Summary  of  analyses  of  wheat  globulin. 


23- 

24. 

25. 

26. 

27. 

Average. 

Carbon 

P.  ct. 
cj  o7 

P.ct. 
ci  oi 

P.ct. 
"\I.OO 

P.ct. 

P.ct. 

P.ct. 
51.  o* 

6  75 

6  Q7 

6.81 

6.85 

Nitrogen  

18.27 
) 

18.48 
(     o  71 

18.26 

0.66 

18.64 

18.29 

18.39 
0.69 

\  23.91 

I   22  Si 

21.26; 

2^.04 

IOO  OO 

IOO  OO 

IOO  OO 

IOO.OO 

teller  (Bull.  42,  Ark.  Agr.  Exp.  Sta.,  1896)  found  a  much  larger  quantity  of  leucosin 
and  globulin  nitrogen  in  the  samples  of  bran  which  he  examined  than  he  found  in  the 
flours. 


4Q  THE;  PROTEINS  OF  THE  WHEAT  KERNEL,. 

THE    PROTEINS    OF    THE   WHEAT   EMBRYO    SOLUBLE   IN  SODIUM- 
CHLORIDE   SOLUTION. 

THE  GI^OBUWN  OF  THE  WHEAT  EMBRYO. 

Wheat-germ  meal  treated  with  10  per  cent  sodium-chloride  brine  forms  a 
dense  jelly-like  mass,  from  which  it  is  nearly  impossible  to  separate  the 
solution. 

With  3  per  cent  brine  a  manageable  extract  can  be  made  by  using  from 
six  to  ten  times  as  much  solvent  as  meal.  Thus  100  grams  of  the  meal 
treated  with  600  cc.  of  3  per  cent  sodium  chloride  yielded  in  1 5  hours  400  cc. 
of  clear  filtrate.  As  has  just  been  shown,  the  aqueous  extract  on  dialysis, 
in  consequence  of  a  change  which  affects  leucosin,  deposits  a  large  amount 
of  protein,  chiefly  in  the  coagulated  form.  In  order  to  obtain  preparations 
of  the  protein  substance  soluble  in  salt  solutions,  but  insoluble  in  water, 
which  should  be  free  from  this  coagulable  albumin,  2000  grams  of  germ 
meal  were  treated  with  20  liters  of  3  per  cent  sodium-chloride  solution 
heated  to  70°,  whereby  the  leucosin  was  coagulated  and  the  salt-soluble 
globulin  brought  into  solution.  The  extract,  neutral  to  litmus,  was  filtered 
clear,  at  once  saturated  with  ammonium  sulphate,  and  the  proteins  thus 
precipitated  collected  on  a  filter,  dissolved  in  water,  and  the  clear  solution 
dialyzed  in  running  water. 

Protein  matter  separated  on  dialysis  in  spheroids,  which,  like  many 
other  plant-globulins,  united  to  a  plastic  mass  on  the  bottom  of  the  dia- 
lyzer.  This  precipitate  was  dissolved  in  sodium-chloride  solution  and,  after 
filtering  absolutely  clear,  dialyzed  for  48  hours,  the  large  precipitate  which 
separated  allowed  to  settle,  and  the  solution,  which  was  nearly  free  from 
protein,  decanted. 

A  portion  of  the  precipitate  was  washed  first  with  water,  which  rendered 
it  opaque  and  dense,  then  with  dilute  and  finally  absolute  alcohol,  and  dried 
over  sulphuric  acid.  This  weighed  5.22  grams,  and  is  preparation  28.  The 
rest  of  the  precipitate  was  completely  dissolved  in  125  cc.  of  10  per  cent 
sodium-chloride  solution.  To  this,  water  was  added  until  its  volume  was 
425  cc. ,  thus  making  a  sodium-chloride  solution  of  nearly  3  per  cent.  From 
this  diluted  solution  a  gummy  deposit  separated,  from  which  the  fluid  was 
soon  completely  decanted.  The  latter  was  further  diluted  with  325  cc.  of 
water  and  the  precipitate  which  resulted  allowed  to  settle  to  a  viscid  trans- 
parent deposit.  From  this  precipitate  the  solution  was  again  decanted  and 
dialyzed  for  48  hours,  but  not  more  than  a  trace  of  globulin  was  deposited. 
The  two  precipitates  produced  by  dilution  formed  preparations  29  and  30, 
weighing  respectively  11.4  grams  and  8.15  grams.  A  part  of  each  of  these 
preparations  was  set  aside  for  analysis,  and  the  rest,  dissolved  together  in  10 
per  cent  sodium-chloride  solution,  allowed  to  stand  over  night  at  4°.  The 


EXPERIMENTAL.  41 

solution  was  then  decanted  from  a  slight  sediment,  filtered  clear,  and  heated 
to  80°,  in  order  to  coagulate  any  leucosin  which  might  be  present,  and  after 
2  hours  filtered  from  a  very  small  coagulum  which  had  gradually  formed. 
This  filtrate  was  dialyzed  in  water  for  4  days,  and  the  globulin  which  sepa- 
rated gave  preparation  31.  The  solution  filtered  from  the  first  dialysis 
precipitates,  which  yielded  preparations  28,  29,  and  30,  was  further  dialyzed  ; 
a  little  globulin,  which  separated,  was  filtered  out  and  the  filtrate  dialyzed 
in  alcohol  for  4  days.  A  precipitate  was  produced  which,  when  washed 
with  absolute  alcohol  and  dried,  weighed  25  grams.  This  substance  con- 
sisted of  protein  which  is  described  on  page  47. 

Another  series  of  fractional  precipitations  of  this  globulin-like  protein 
was  made  by  extracting  4  kilograms  of   the  oil-free  germ  meal   with  27 
liters  of  3  per  cent  sodium-chloride  solution  heated  to  67°    at   the   time 
it   was   applied   to  the  meal.     The  mixture  was   thoroughly   stirred   and 
thrown  on  filters.     A  clear  filtrate  of  about  12  liters  was  finally  obtained, 
which  was  saturated  with  ammonium  sulphate.     The  precipitate  produced 
was  dissolved  in  water  and   its  solution   dialyzed   for  48  hours ;   where- 
upon a  large  quantity  of  spheroids  separated,  which  on   settling  united 
to  a  coherent  mass.     This  precipitate  was  washed  by  decantation  with 
water,  dissolved  in  brine,  and  its  solution  made  faintly  alkaline  to  litmus  by 
cautiously  adding  decinormal  potassium-hydroxide  solution.     In  order  to 
separate  phosphoric  acid,  a  little  calcium-chloride  solution  was  then  added 
to  this  very  slightly  alkaline  liquid,  and  the  latter,  though  apparently  free 
from  any  precipitate  of  calcium  phosphate,  was  filtered,  whereby  a  little 
suspended  matter  was  removed.     The  solution  was  made  exactly  neutral  to 
litmus  by  adding  56  cc.  decinormal  hydrochloric  acid  and  dialyzed  for  18 
hours.     A  gummy  precipitate  (A)  adhering  to  the  bottom  of  the  dialyzer 
then  separated,  from  which  the  solution  (B)  was  decanted  almost  completely. 
The  precipitate  (A)  was  dissolved  in  about  200  cc.  of  5  per  cent  sodium- 
chloride  solution  and  the  liquid  was  poured  into  800  cc.  of  water.     The 
resulting  flocculent  precipitate  settled  rapidly  to  a  coherent  deposit,  from 
which  the  solution  was  decanted.     The  deposit  was  repeatedly  washed  by 
decantation  with  water,  which  caused  it  to  lose  its  gummy  character  and 
become  opaque,  white,   and  granular.     It  weighed   15.5  grams  and  was 
preparation  32.     The  solution  marked  B  was  further  dialyzed  for  48  hours, 
when  a  second   precipitate    formed,  which,  like   32,  completely  dissolved 
in  sodium-chloride  solution  to  a  solution  perfectly  neutral  to  litmus.     This 
precipitate   was  washed  by   decantation   with   water,    but   the  finer  part 
settled  so  slowly  that  it  was  necessary  to  decant  it  together  with  the  water. 
The  sediment,  after  exhausting  with  absolute  alcohol  and  drying,  weighed 
23-5  grams,  and  formed  preparation  33.     On  long  standing  the  decanted 
washings  deposited  the  finely  divided  matter,  which  was  then  collected  on  a 


42  THE;  PROTEINS  OF  THE;  WHEAT  KERNEX. 

filter,  dissolved  in  sodium- chloride  solution,  and  precipitated  by  water,  giving 
15.4  grams  of  preparation  34. 

To  determine  the  quantity  of  globulin  contained  in  the  oil-free  germ  meal, 
200  grams  of  the  meal  were  treated  with  2000  cc.  of  3  per  cent  sodium- 
chloride  solution  heated  to  65°  and  the  extract  filtered  perfectly  clear.  Of 
this,  1000  cc.  were  dialyzed  until  free  from  chlorides,  when  the  precipitate  of 
spheroids  was  filtered  out.  This  preparation,  35,  formed  5.05  per  cent  of 
the  oil-free  meal. 

To  obtain  a  quantity  of  this  globulin  for  digestion  with  pepsin,  a  quantity 
of  germ  meal  was  extracted  with  3  per  cent  sodium-chloride  solution  heated 
to  70°;  the  extract  was  filtered  clear  and  saturated  with  ammonium  sul- 
phate. The  precipitate  produced  was  dissolved  in  water  and  the  resulting 
gummy  and  somewhat  turbid  solution  filtered  clear.  The  filtrate  was  dia- 
lyzed until  the  solution  gave  no  turbidity  on  pouring  into  distilled  water. 
The  protein,  which  had  then  separated  in  spheroids,  weighed  27.3  grams, 
preparation  36. 

A  part  of  the  extract  from  which  36  had  been  prepared  was  mixed  with  an 
equal  volume  of  decinormal  potassium  hydroxide  solution — about  twice  the 
quantity  necessary  to  neutralize  the  extract  to  phenolphthalein.  The  solu- 
tion was  then  dialyzed  in  distilled  water  frequently  renewed,  and  in  this  way  a 
considerable  quantity  of  phosphorus  was  separated  in  the  alkaline  dialysate. 
When  all,  or  nearly  all,  which  it  was  possible  to  separate  in  this  way  had 
been  removed,  the  solution  in  the  dialyzer  was  neutralized  with  hydrochloric 
acid  until  it  no  longer  reacted  alkaline  to  litmus.  This  caused  a  turbidity. 
The  acid  was  then  further  added  until  an  acid  reaction  with  litmus  was 
obtained,  producing  a  precipitate  from  which,  after  settling,  the  solution  was 
decanted.  The  precipitate  was  then  dissolved  in  sodium-chloride  solution, 
its -solution  filtered  clear  and  dialyzed,  whereby  a  substance  was  precipitated 
in  spheroids,  which  formed  preparation  37,  weighing  3.0  grams.  These 
preparations  had  the  composition  shown  in  table  7. 

TABLE  7. — Composition  of  preparations  extracted  by  sodium-chloride  solution 
from  the  wheat  embryo. 


28. 

29. 

30- 

Si- 

32. 

33- 

34- 

35- 

36. 

37- 

Carbon  

P.ct. 

P.ct. 

P.ct. 

48.77 

P.ct. 
5,0.  o* 

P.ct. 

CO.  2^ 

P.ct. 
48.17 

P.ct. 

4Q.  70 

P.ct. 

48.  7<5 

P.ct. 
4Q.7Q 

P.ct. 

48.67 

Hydrogen  

6  Ad 

7.04 

6.8q 

6.S4 

6.78 

6.52 

6.76 

6.56 

Nitrogen  .  .       

18.14 

18.21 

18  12 

18  T.Q 

lS.2^ 

18  06 

17.  QC 

18.16 

18.01 

I7.Q7 

Sulphur  

0.4.0 

o.  56 

O.CT 

0.60 

o.s^ 

O.SS 

0.48 

0.63 

0.61 

0.61 

Phosphorus  

1.15 

i.oi. 

1.^5 

0.76 

0.56 

1.41 

1.17 

1.41 

I.  ii 

1.55 

Ash  

2.  20 

1.86 

2.2S 

i.V> 

1.22 

3.85 

2.60 

2.66 

I.  II 

2.  04 

P2O5  in  ash  

1.66 

1.14 

1.68 

0.84 

0.80 

2.OO 

1.82 

2.OO 

0.68 

2.0.O 

EXPERI  MENTAL. 


43 


These  analyses,  when  calculated  free  from  nucleic  acid  and  ash,  as  was 
done  for  the  albumin  preparations,  in  the  manner  described  on  page  27,  gave 
the  results  set  out  in  table  8. 

TABI/E  8. — Composition  of  the  globulin  contained  in  the  preparations  extracted 
from  the  wheat  embryo  by  sodium-chloride  solution. 


Carbon  

28. 

29. 

30. 

31. 

32. 

33- 

34- 

35- 

36. 

37- 

P.ct. 

P.ct. 

P.ct. 

51-37 
6.83 
18.62 

0.60 

22.58 

P.ct. 

51.58 

7-31 
18.70 

0.66 
21-75 

P.ct. 

51.40 
7.08 

18.45 
0.57 
22.50 

P.ct. 
5I-56 
7.07 
18.85 
0.67 
21.85 

P.ct. 

51-86 
7.19 
18.41 

0-55 
21.99 

P.ct. 

51.40 
6.94 
18.71 
0.75 

22.20 

P.ct. 

51.98 
7.12 

18.37 

0.70 

21.83 

P.ct. 

51.70 
7.05 
18.53 

0-75 
21.97 

Hydrogen  .  . 
Nitrogen  .  .  . 
Sulphur  — 
Oxygen  .... 

18.59 
0-57 

18.59 
0.63 

IOO.OO 

IOO.CO 

IOO.OO 

IOO.OO 

IOO.OO 

IOO.OO 

IOO.OO 

IOO.OO 

These  figures  plainly  indicate  that  these  globulin  preparations  are  mixtures 
of  nucleates  of  protein  substance  of  the  same  ultimate  composition,  and  con- 
tain from  5  to  15  per  cent  of  nucleic  acid.  This  protein  has  very  nearly  the 
same  composition  as  the  globulin  occurring  in  the  kernel  of  wheat,  rye, 
barley,  and  maize.  In  the  entire  kernel  so  little  of  this  globulin  is  present 
that  it  is  difficult  to  prepare  it  pure  therefrom.  For  this  reason  the  analyses 
given  in  table  9  do  not  agree  as  closely  as  they  might  otherwise  be  expected 
to.  From  the  whole  seed  this  globulin  is  obtained  entirely  free  from  phos- 
phorus, which  is  probably  due  to  the  much  greater  proportion  of  protein 
matter  to  nucleic  acid  in  the  entire  seed  compared  with  that  existing  in  the 
embryo. 

TABI,E  9. — Composition  of  the  globulin  contained  in  various  cereals. 


Wheat 
embryo. 

Wheat 
kernel. 

Rye1 
kernel. 

Maize2 
kernel. 

Barley3 
kernel. 

Carbon  

P.ct. 
SI.">7 

P.  ct. 
ci.o^ 

P.ct. 
si.  iq 

P.ct. 

SI.QQ 

P.ct. 
50  88 

Hydrogen  

7.O7 

6.85 

6.74 

6.81 

6.65 

18.60 

18.^9 

18.19 

18  02 

18  10 

Sulphur  

o  6s 

o  65 

f      o  66 

1           ".uw 

1 

Oxygen  

22.11 

2^.08 

}    2388 

I    22.52 

}    24.37 

IOO.OO 

IOO.OO 

IOO.OO 

IOO.OO 

IOO.OO 

1  Osborne,  Journal  American  Chemical  Society,  1895,  xvii,  p.  429. 

2  Chittenden  &  Osborne,  American  Chemical  Journal,  1891,  xiu,  pp.  327,  385, 

and  1892,  xiv,  p.  20. 

3  Osborne,  Journal  American  Chemical  Society,  1895,  xvn,  p.  539. 


44 


THE    PROTEINS    OF   THE    WHEAT    KERNEL. 


Having  determined  the  composition  of  this  globulin-like  protein  and  also 
that  of  the  albumin,  it  became  clear  that  several  preparations  obtained  from 
the  aqueous  extract  were  mixtures  of  these  two  substances,  thus  showing 
the  globulin  to  be  present  to  some  extent  in  the  aqueous  extract. 

As  noted  on  page  24,  when  2000  cc.  of  an  aqueous  extract  of  about  650 
grams  of  the  meal  were  dialyzed  in  running  water  for  4  days  a  dense  tur- 
bidity was  formed,  which  could  not  be  removed  by  filtration.  This,  however, 
on  adding  a  little  hydrochloric  acid,  was  converted  into  a  precipitate,  which 
was  readily  dissolved  by  adding  sodium  chloride  sufficient  to  make  a  3  per 
cent  solution,  and  was  precipitated  from  this  solution  by  dialysis. 

Preparation  38,  weighing  9  grams,  was  thus  obtained,  which,  dried  at  1 10° , 
had  a  composition  which  corresponds  pretty  nearly  with  that  of  a  mixture  of 
60  per  cent  of  the  globulin  with  40  per  cent  of  leucosin,  except  that  the 
amount  of  sulphur  found  was  somewhat  greater  than  that  calculated.  The 
analysis  is  shown  in  the  following  table  : 

Preparations  38  and  jp. 


Preparation  38. 

Preparation  39. 

I. 

ii. 

Aver- 
age. 

Corrected 
for  ash 
and  nu- 
cleic acid. 

Calculated 
for  60  per 
cent  globu- 
lin and  40 
per  cent 
leucosin. 

I. 

Corrected 
for  ash 
and  nu- 
cleic acid. 

Calculated 
for  40  per 
cent  globu- 
lin and  60 
per  cent 
leucosin. 

Carbon  

P.  ct. 
48.30 

6.49 
17.40 
0.83 

I.QI 

P.ct. 

47.92 
6.41 

17.24 
0.85 

P.ct. 

48.11 

6-45 
17.32 
0.84 

I.QI 

P.ct. 
5I-70 
7.07 

17-74 

1.08 

P.ct. 

51-95 
7.07 

17-74 
0.91 

P.ct. 

49-49 
6.81 
16.87 

0-93 
0.89 

P.ct. 
51.80 
7.14 
17.32 
I.I4 

P.ct. 
52.13 
7-03 
17.30 
1.05 

Hydrogen  
Nitrogen  

Sulphur  

Phosphorus.  .  .  . 

Oxvtren 

22.51 

22.53 

22.6O 

22.49 

Ash  

VQ'i 

4.00 

PoO5  in  ash  .... 

2.  Q6; 

2.OI 

IOO.OO 

IOO.OO 

IOO.OO 

IOO.OO 

After  heating  another  portion  of  the  same  aqueous  extract  to  65°  for  some 
time  and  filtering  off  the  coagulum,  the  filtrate  was  dialyzed  for  5  days  into 
alcohol  and  the  precipitate  thereby  produced  filtered  out  and  exhausted  with 
water.  The  residue  of  protein  matter  coagulated  by  alcohol,  weighing  6.7 
grams  and  marked  preparation  39,  was  then  dried  at  no0  and  analyzed  with 
the  results  shown  in  the  preceding  table. 

This  analysis  corresponds  quite  nearly  with  that  of  a  mixture  of  40  per 
cent  of  the  globulin  with  60  per  cent  of  leucosin. 


EXPERIMENTAL.  45 

No  earlier  analyses  of  the  wheat  globulin  are  on  record.  Weyl1  was  the 
first  to  call  attention  to  the  presence  of  globulin  in  wheat,  and  says  that 
"  besides  vegetable- vitellin  I  detected  in  the  10  per  cent  sodium-chloride 
extract  of  the  pulverized  seeds  of  wheat,  peas,  oats,  white  mustard,  and 
sweet  almonds  a  second  globulin  substance. ' '  This  he  calls  vegetable-myosin 
and  gives  its  coagulation-point  at  55°  to  60°.  It  is  probable  that  Weyl  in 
some  way  mistook  for  a  globulin  the  albumin  already  described. 

Later,  Weyl  &  Bischoff 2  state  : 

On  investigating  the  proteins  of  wheat,  one  of  us  found  chiefly  an  albuminous  sub- 
stance which,  on  account  of  its  resemblance  to  myosin,  was  named  vegetable-myosin. 
This  vegetable-myosin  must  be  the  mother-substance  of  the  gluten,  since  in  wheat  meal, 
together  with  it,  other  nitrogenous  substances  exist,  at  the  most,  only  in  very  small 
amount. 

On  what  experimental  evidence  this  statement  rests  the  writer  has  been 
unable  to  discover,  and  in  view  of  his  experience  he  is  at  a  loss  to  under- 
stand it. 

Martin  *  considers  wheat  flour  to  contain  a  large  amount  of  globulin  of  the 
myosin  type,  coagulating  between  55°  and  60°,  precipitated  by  saturation 
with  sodium  chloride  and  ammonium  sulphate.  Here  again  the  small 
quantity  of  albumin  contained  in  the  flour  has  evidently  been  mistaken 
for  a  large  quantity  of  vegetable-myosin.  This  perhaps  is  not  surprising, 
as  the  precipitates  obtained  by  saturating  sodium-chloride  extracts  with 
ammonium  sulphate  appear  very  bulky,  and  in  the  absence  of  an  actual 
determination  of  the  weight  of  these  precipitates  misleading  conclusions 
might  easily  be  reached.  The  only  globulin  found  by  the  writer  in  extracts 
of  wheat  meal,  either  winter  or  spring  wheat,  is  the  one  just  described, 
which  in  properties  and  composition  closely  resembles  those  globulins  which 
have  been  found  in  other  seeds. 

THE  PROTEOSE   OF  WHEAT   FLOUR. 

As  already  stated  in  describing  the  reaction  of  the  extract  freed  from 
globulin  by  dialysis,  there  was  found  in  it  one  or  more  proteoses,  besides 
the  albumin  just  described.  These  were  almost  wholly  precipitated  by 
saturation  with  sodium  chloride  or  by  adding  20  per  cent  of  this  salt  to  the 
solution,  together  with  a  little  acetic  acid. 

If  the  albumin  is  completely  removed  by  heat  and  the  filtered  solution 
then  concentrated,  a  coagulum  gradually  develops.  This  substance  must 
be  derived  from  the  proteose-like  protein,  as  this  forms  nearly  if  not  quite 
all  the  protein  substance  remaining  in  solution  before  concentration.  If 

1  Weyl,  Zeitschrift  fur  physiologische  Chemie,  1877,  I,  p.  72. 

2  Weyl  &  Bischoff,  Berichte  der  deutschen  chemischen  Gesellschaft,  1880,  xm,  p.  367. 

3  Martin,  British  Medical  Journal,  1886,  n,  p.  104. 


46 


THE    PROTEINS    OF   THE    WHEAT    KERNEL. 


the  coagulum  is  removed  by  concentration  and  long-continued  heating  and 
subsequent  filtration,  wholly  uncoagulable  proteose-like  substances  are 
found  in  solution.  The  amount  of  proteose  is  extremely  small,  and  no 
preparations  were  made  for  analysis.  The  coagula  obtained  by  concen- 
trating the  solution  filtered  from  preparations  3  and  4,  respectively,  were 
analyzed  with  the  results  shown  in  the  accompanying  table  under  the  head 
"Preparation  40." 

Another  coagulum  similarly  obtained  from  the  solution  filtered  from  prep- 
aration 4  was  also  analyzed  and  the  following  figures  obtained  : 

Preparations  40  and  41. 


Preparation  40. 

Preparation  41. 

I. 

II. 

Average. 

Ash-free. 

I. 

II. 

Average. 

Ash-free. 

Carbon  

P.  ct. 
51-35 

P.ct. 
51.60 

P.  et. 

51.48 

P.ct. 
51.62 

P.ct. 

51-73 
6.82 

P.ct. 

P.  ct. 

51-73 
6.82 
17.28 

P.ct. 
51-86 
6.82 
17.32 

24.00 

Hydrogen  .  .  . 

Nitrogen  .... 
Sulphur  

16.79 

16.58 

16.69 

16.73 

17.29 

17.26 

Oxygen  

).... 
0.25 

0.25 

Ash  

0.27 





100.00 

THE   PROTEOSE   OF   THE   WHEAT   EMBRYO. 

In  making  the  preparations  from  the  embryo  already  described  consider- 
able quantities  of  crude  proteose  were  obtained  from  both  the  aqueous  and 
sodium-chloride  extracts.  After  the  leucosin  and  the  globulin  had  been 
separated  as  completely  as  possible,  the  solutions  containing  the  proteoses 
were  dialyzed  in  alcohol,  and  the  precipitates  produced  were  washed  and 
dried  over  sulphuric  acid. 

A  mixture  weighing  15.4  grams  was  made  by  uniting  several  such  prep- 
arations that  had  been  obtained  from  aqueous  extracts  from  which  most  of 
the  other  proteins  had  been  separated,  without  heat,  by  saturating  with 
sodium  chloride  and  dialysis  in  alcohol.  The  mixture  contained  much 
matter  made  insoluble  in  water  by  the  final  treatment  with  alcohol.  This 
was  filtered  out,  washed  thoroughly  with  water  and  with  alcohol,  and  when 
dried  weighed  4.18  grams,  and  was  marked  preparation  42.  The  filtrate 
from  this  was  saturated  with  ammonium  sulphate,  the  precipitate  redissolved> 
and  again  precipitated  in  the  same  way.  The  solution  of  the  second  pre- 
cipitate was  dialyzed  in  cold  distilled  water  until  free  from  sulphate,  and  then 
for  several  days  in  alcohol ;  the  precipitate  thus  produced  was  dissolved  in 
water,  a  little  insoluble  matter  filtered  out,  and  its  clear  solution  saturated 


EXPERIMENTAL.          xg;cuLTUX  47 


with  sodium  chloride,  which  produced  a  small  precipitate.  This  was  filtered 
out,  dissolved,  and  its  solution  dialyzed  in  water.  The  salt-saturated  filtrate 
was  likewise  dialyzed,  and  when  both  solutions  were  free  from  chlorine  the 
dialyzers  were  transferred  to  alcohol  and  the  proteose  thereby  precipitated. 
The  proteose  separating  on  saturation  with  salt  gave  o.  6  gram  of  prepara- 
tion 43  ;  that  from  the  salt-saturated  solution  0.97  gram  of  preparation  44. 
This  small  yield  of  proteose  indicates  that  the  greater  part  had  diffused 
through  the  parchment  paper  during  the  long  dialysis  to  which  the  solutions 
had  been  subjected. 

Another  crude  product  was  obtained  by  dialyzing  an  aqueous  extract  in 
alcohol  after  separating  the  leucosin  which  had  been  coagulated  by  heat. 
This,  weighing  35  grams,  was  dissolved  in  water  and  the  insoluble  matter 
filtered  out,  washed  and  dried,  giving  preparation  45,  weighing  7.26  grams. 

The  filtered  solution  was  saturated  with  ammonium  sulphate,  the  pre- 
cipitate dissolved  in  water,  and  the  clear  solution  dialyzed  in  distilled  water 
until  free  from  sulphates,  and  then  in  alcohol.  The  substance  thus  sep- 
arated was  again  dissolved  in  water  and  its  solution  saturated  with  salt  ;  the 
precipitate  thus  produced  was  dissolved  in  water,  and  its  solution,  as  well  as 
the  salt-saturated  filtrate,  were  dialyzed  in  water.  When  free  from  chlorine, 
these  solutions  were  dialyzed  in  alcohol  and  yielded,  respectively,  prepara- 
tions 46,  weighing  4  grams,  and  47,  weighing  1.84  grams. 

Another  preparation  of  crude  proteose  was  obtained  by  extracting  the 
meal  as  described  on  page  41  with  3  percent  sodium-chloride  solution  heated 
to  70°,  dialyzing  the  extract  in  water,  coagulating  the  leucosin  by  heat,  and 
precipitating  the  proteose  by  dialysis  in  alcohol.  A  mixture  of  such  prep- 
arations, weighing  31.6  grams,  was  treated  with  water,  the  insoluble  matter 
filtered  out,  washed,  and  dried,  giving  5.16  grams  of  preparation  48. 

The  filtered  solution  was  saturated  with  ammonium  sulphate,  the  precipi- 
tate dissolved  in  water,  the  solution  dialyzed  in  distilled  water  till  free  from 
sulphate,  and  then  in  alcohol.  The  separated  proteose  was  redissolved  in 
water  and  its  solution  saturated  with  sodium  chloride.  The  precipitate 
which  resulted  was  filtered  out,  dissolved  in  water,  and  its  solution,  as  well 
as  the  salt-saturated  filtrate,  were  dialyzed  in  water  till  free  from  chlorine, 
and  finally  in  alcohol. 

The  products  thus  obtained  formed,  respectively,  preparation  49,  weigh- 
ing 0.75  gram,  and  50,  weighing  1.35  grams.  One  other  proteose  prepara- 
tion was  made  from  the  aqueous  extract  described  on  page  26,  from  which 
the  phosphorus  was  largely  separated  by  making  it  slightly  alkaline  and 
adding  calcium  chloride.  After  heating  the  extract  to  boiling  and  filtering 
out  the  coagulum,  the  filtrate  was  dialyzed  in  alcohol,  the  resulting  pre- 
cipitate dehydrated  with  absolute  alcohol,  dried  over  sulphuric  acid,  redis- 
solved in  water,  and  precipitated  by  saturating  with  ammonium  sulphate. 


48 


THE)    PROTEINS    OF   THE    WHEAT    KERNEL. 


The  gummy  precipitate,  having  the  general  appearance  and  properties  of 
similar  precipitates  of  the  proteoses  obtained  by  the  action  of  pepsin,  was 
dissolved  in  water,  dialyzed  free  from  sulphates,  and  then  precipitated  by 
dialysis  in  alcohol,  giving  2  grams  of  preparation  51.  These  preparations 
were  dried  at  110°  and  analyzed  with  the  results  shown  in  table  10. 

TABI,E  10. — Composition  of  alcohol  coagula  and  of  protease  prepared  from 

the  wheat  embryo. 


Residues  of  other  pro- 
teins coagulated  by 
alcohol. 

Proteose  precipitated 
by  sodium  chloride. 

Proteose  soluble  in  saturated 
NaCl  solution. 

42. 

45- 

48. 

43- 

46. 

49- 

44- 

47- 

50. 

51. 

Carbon  

P.ct. 
52.36 
6.98 
16.01 
1.85 
22.80 

P.ct. 
49-44 
6.85 
16.00 
4.08 
23-63 

P.ct. 

5i  93 
6.87 
16.30 
1.30 
23.60 

P.ct. 

P.ct. 
49-94 
6.80 

P.ct. 

P.ct. 

48.46 
6.70 
16.91 

}  27.93 

P.ct. 

48.70 

6.73 
16.76 

27.81 

P.ct. 

48.44 

6.71 

16.16 
28.69 

P.  ct. 

48.99 
6.85 
16.89 

(      1.  10 

1  26.17 

IOO.OO 

1.27 

Hydrogen  

Nitrogen  

16.79 

17.08 
1.24 
24.94 

16.26 

Sulphur  

Oxygen  .  . 

Ash  

IOO.OO 

0.81 

IOO.OO 

14-13 

IOO.OO 

o.95 



IOO.OO 

0.30 

0.77 

IOO.OO 

1-13 

IOO.OO 
1.  00 

IOO.OO 

0.74 

From  these  analyses  it  is  seen  that  the  matter  insoluble  in  water,  forming 
preparations  42,  45,  and  48,  consists  of  coagulated  protein  apparently  mostly 
derived  from  leucosin.  The  high  proportion  of  sulphur  in  42  and  45  is  due 
to  calcium  sulphate  precipitated  by  alcohol  from  the  aqueous  extract.  The 
remaining  preparations  have  the  low  percentage  of  carbon  characteristic  of 
proteoses  made  by  pepsin  digestion. 

THE   PROPORTIONS  OP  THE   VARIOUS   PROTEIN  SUBSTANCES   OP 
THE  WHEAT   EMBRYO. 

Twenty  grams  of  fresh  germ  meal,  from  which  the  ether- soluble  constit- 
uents had  not  been  separated,  were  treated  with  500  cc.  of  water,  and  after 
shaking  for  some  time  the  extract  was  filtered  clear.  Two  portions  of  100 
cc.  each  were  treated  with  a  few  drops  of  very  dilute  hydrochloric  acid  and 
heated  in  a  boiling  water-bath.  The  coagulum  which  separated  was  col- 
lected on  a  filter  and  its  nitrogen  determined.  To  the  filtrate  from  one 
coagulum  tannin  was  added,  and  nitrogen  was  determined  both  in  the  pre- 
cipitate and  in  the  filtrate.  Another  lot  of  20  grams  was  treated  in  the  same 
way  and  nitrogen  determined  in  the  heat-coagulum  formed  in  each  of  two 
portions  of  100  cc.  The  amount  of  nitrogen  corresponding  to  i  gram  of 
germ  meal  was  found  in  the  four  coagula  to  be  0.0163  gram,  0.0156  gram, 
0.0159  gram,  and  0.0162  gram,  in  the  tannin  precipitate  0.0062  gram,  and  in 
the  solution  filtered  from  the  latter  0.0062  gram. 


EXPERIMENTAL.  49 

Twenty  grams  of  germ  meal  were  extracted  with  500  cc.  of  3  per  cent 
sodium-chloride  solution  heated  to  70°,  whereby  the  leucosin  was  coagu- 
lated and  the  globulin  and  proteose  dissolved.  Of  the  clear  filtered  extract 
100  cc.  yielded  with  tannin  a  precipitate  containing  0.0166  gram  nitrogen 
per  gram  of  meal  extracted. 

Two  portions  of  the  meal,  each  of  i  gram,  were  exhausted  with  3  per 
cent  sodium-chloride  solution  heated  to  70°  and  nitrogen  determined  in  the 
residues.  The  0.0331  gram  and  0.0309  gram  of  nitrogen  found  in  the  resi- 
dues were  from  the  leucosin  and  insoluble  nitrogenous  bodies,  so  that  the 
nitrogen  belonging  to  the  latter  equaled  0.0171  gram  and  0.0149  gram. 
From  the  average  of  these  figures  we  find  the  following  amounts  of  the 
different  forms  of  nitrogen  in  i  gram  of  the  wheat-germ  meal : 

N,  grains. 

Insoluble  in  water  and  salt  solution 0160 

Insoluble  in  water,  but  soluble  in  salt  solution  (globulin  nitrogen) . .       .0100 

Soluble  in  water  and  coagulable  by  heat  (albumin  nitrogen) 0160 

Soluble  in  water,  uncoagulable  by  heat,  precipitable  by  tannin  (pro- 
teose nitrogen) 0050 

Not  precipitable  by  tannin  (non-protein  nitrogen) 0060 

Total 0530 

Found  by  direct  nitrogen  determination 0531 

It  has  been  shown  that  the  coagulated  leucosin  preparations  contain  about 
10  per  cent  of  nucleic  acid,  the  globulin  about  15  per  cent,  while  those  of 
the  proteose  contain  none.  Deducting  these  quantities  from  the  nitrogen 
given  above,  it  is  found  that  9.5  per  cent  of  the  embryo  is  leucosin,  4.84  per 
cent  globulin,  and  3.03  per  cent  proteose. 

The  bodies  which  are  represented  by  the  insoluble  nitrogen  could  not  be 
separated  from  the  embryo.  The  residue,  after  extraction  with  hot-salt 
solution,  contained  0.0076  gram  of  phosphorus.  In  view  of  the  large 
proportion  of  nucleic  acid  found  in  the  extracts  of  the  embryo,  it  is  not 
improbable  that  this  phosphorus  mostly  belongs  to  nucleic  acid,  and  that  the 
insoluble  nitrogen  largely  belongs  to  compounds  of  protein  with  relatively 
much  nucleic  acid. 

DIGESTION   OF  THE   PHOSPHORUS-CONTAINING   PROTEIN   PREPARATIONS 

WITH  PEPSIN-HYDROCHLORIC  ACID. 

LEUCOSIN  NUCLEATE. 

Ten  grams  of  the  coagulated  albumin,  preparation  8,  were  suspended  in 
400  cc.  of  water  and  dissolved  by  adding  100  cc.  of  decinormal  potassium- 
hydroxide  solution.  To  the  nearly  clear  solution  which  resulted  an  equal 
volume  of  0.4  per  cent  hydrochloric  acid  was  added,  together  with  some 
pepsin,  and  the  mixture  digested  at  37°.  In  a  short  time  the  solution 
4 


5°  THE  PROTEINS  OF  THE  WHEAT  KERNEL. 

became  perfectly  clear,  but  later  deposited  a  large  coherent  precipitate,  which 
gradually  contracted,  but  at  the  same  time  retained  the  form  of  the  lower 
part  of  the  beaker.  From  this  the  clear  solution  was  decanted,  the  precip- 
itate thoroughly  washed  by  decantation,  suspended  in  water  and  dissolved 
by  adding  28  cc.  of  decinormal  potassium-hydroxide  solution,  an  amount  of 
alkali  just  sufficient  to  dissolve  all  the  substance,  and  at  the  same  time  make 
the  solution  neutral  to  litmus.  When  to  this  solution  decinormal  acid  was 
gradually  added,  no  precipitate  appeared  until  nearly  one-half  the  quantity 
of  acid  required  for  complete  neutralization  had  been  added,  but  with  28  cc. 
the  solution  was  neutralized  and  the  nuclein  completely  precipitated,  the 
addition  of  2  cc.  more  acid  giving  no  turbidity  in  the  filtered  solution.  This 
precipitate  formed  preparation  52,  weighing  1.54  grams. 

To  precipitate  this  substance  a  quantity  of  acid  was  added  exceeding  that 
of  the  alkali  employed  for  solution  by  just  2  cc.  The  filtrate  from  the  pre- 
cipitate, however,  required  not  2  cc.  of  alkali,  but  8.5  cc.  for  neutralization 
to  phenolphthalein,  showing  6.5  cc.  of  alkali  to  have  been  neutralized  by 
the  acid  of  the  nuclein  originally  dissolved.  The  neutralized  filtrate  left  on 
evaporation  0.3975  gram  of  substance,  the  aqueous  solution  of  which  was 
precipitated  by  hydrochloric  or  nitric  acid,  but  not  by  ammonium  molybdate 
solution  until  after  boiling  with  acid  for  some  little  time,  when  yellow  phos- 
phomolybdate  was  precipitated.  These  facts  indicate  the  presence  in  this 
filtrate  of  a  nucleic  acid. 

More  nuclein  was  made  from  the  same  preparation,  7,  by  suspending  30 
grams  in  0.2  per  cent  hydrochloric  acid  containing  pepsin,  which,  even  at 
20°,  caused  within  2  hours  complete  solution  of  the  coagulated  protein. 
The  solution  was  digested  at  37°  for  48  hours,  during  which  time  much 
nuclein  separated,  having  the  appearance  and  properties  of  the  preparation 
just  described. 

After  decanting  the  clear  solution  and  thoroughly  washing  the  residual 
nuclein,  the  latter  was  suspended  in  water  and  dissolved  in  72  cc.  decinor- 
mal potassium-hydroxide  solution.  The  solution  thus  obtained  was  made 
neutral  to  litmus  by  adding  1 1  cc.  of  decinormal  hydrochloric  acid,  but  no 
precipitate  appeared  till  1.5  cc  more  of  acid  were  added.  To  the  solution 
72  cc.  decinormal  hydrochloric  acid  were  added,  giving  a  precipitate,  prepa- 
ration 53,  which  weighed  3.4  grams.  The  filtrate  from  this  precipitate,  as 
in  the  former  case,  was  strongly  acid,  requiring  12  cc.  of  decinormal  potas- 
sium hydroxide  to  neutralize  it  to  phenolphthalein.  Two  other  preparations 
of  nuclein  were  made  from  8.493  grams  of  13  and  9.804  grams  of  16,  both 
being  substances  precipitated  from  the  aqueous  extract  by  saturating  with 
sodium  chloride.  Bach  portion  was  suspended  in  about  300  cc.  of  0.2  per 
cent  hydrochloric  acid,  containing  o.  i  gram  of  pepsin,  and,  with  frequent 


EXPERIMENTAL.  cj! 

stirring,  digested  at  40°  for  24  hours.  Throughout  the  digestion  a  large 
part  of  the  substance  remained  undissolved.  An  equal  volume  of  0.2  per 
cent  hydrochloric  acid,  containing  o.  i  gram  of  pepsin,  was  again  added  to 
each,  and  the  digestion  continued  for  24  hours  longer.  The  insoluble  matter 
which  remained  was  not  coherent  like  the  two  former  nuclein  products,  but 
consisted  of  a  white,  very  finely  divided  substance,  which  was  easily  filtered 
out  and  washed.  From  13,  4.04  grams  of  preparation  54  were  obtained, 
and  from  16,  4.16  grams  of  55. 

GLOBUWN  NUCLEATE. 

Fifteen  grams  of  a  mixture  of  nearly  equal  parts  of  the  globulin  prepa- 
rations 32  and  33  were  next  suspended  in  0.2  per  cent  hydrochloric  acid 
containing  0.2  gram  of  pepsin,  which,  within  a  short  time,  almost  completely 
dissolved  the  protein  matter.  From  this  solution,  on  further  digestion,  the 
nuclein  separated,  forming  a  coherent  deposit.  After  72  hours'  digestion 
the  clear  solution  was  decanted,  the  deposit  dissolved  in  a  little  ammonia,  and 
its  solution  filtered  perfectly  clear  from  a  very  slight  gelatinous  residue. 
The  solution  was  then  treated  with  acetic  acid  added  in  excess  of  the 
amount  necessary  to  neutralize  it  to  litmus.  Since,  even  on  standing,  the 
precipitate  so  produced  separated  imperfectly,  an  equal  volume  of  alcohol 
was  added.  The  substance,  which  then  separated  well,  gave  2.38  grams  of 
preparation  56,  or  about  16  per  cent  of  the  original  substance. 

The  filtrate  from  the  acetic  acid  precipitate,  on  adding  hydrochloric  acid, 
gave  a  further  slight  precipitate,  which  had  properties  characteristic  of 
nucleic  acid. 

Still  another  preparation  of  nuclein  was  made  from  the  globulin  by  sus- 
pending 10  grams  of  36  in  water  and  adding  50  cc.  of  decinormal  potassium- 
hydroxide  solution.  This  solution  was  neutralized  and  an  equal  volume  of 
0.4  per  cent  hydrochloric  acid  at  once  added,  producing  a  turbid  solution, 
which,  however,  contained  no  visible  particles.  To  this  pepsin  was  added 
and  the  mixture  digested  for  40  hours,  during  which  time  a  coherent  deposit 
of  nuclein  formed  on  the  bottom  of  the  beaker.  From  this  the  clear  solution 
was  decanted.  The  deposit  was  then  thoroughly  washed  with  water  and 
dissolved  in  43  cc.  of  decinormal  potassium-hydroxide  solution.  To  this 
clear  solution  43  cc.  of  decinormal  hydrochloric  acid  were  added,  causing  a 
gummy  precipitate,  which  could  not  be  filtered  until  15  cc.  more  acid  had 
been  added,  when  the  precipitate  rapidly  settled  as  a  coherent  deposit,  from 
which  the  solution  was  soon  decanted.  This  solution  required  for  neutrali- 
zation to  litmus  1 6  cc.  of  decinormal  potassium-hydroxide  solution,  and  to 
phenolphthalein  18  cc.  The  precipitate  when  washed  and  dried  gave  2.2 
grams  of  preparation  57. 


THE    PROTEINS   OF   THE    WHEAT    KERNEL. 


These  six  preparations  were  all  dried  at  1 10°  and  anatyzed  with  the  results 
shown  in  table  1 1 . 

TABLE  u. — Composition  of  nuclein  from  the  proteins  of  the  wheat  embryo. 


52. 

53- 

54- 

55- 

56. 

57- 

Carbon  

P.ct. 
44.87 

P.ct. 
44.^5 

P.ct. 

42.68 

P.ct. 
47.  -is 

P.  ct. 

^Q.42 

P.ct. 
41  Q2 

Hydrogen  
Nitrogen  

5-82 
16.04 

5-77 

16.64 

5-45 
16.  12 

5-47 
16.01 

5-03 

ID.OS 

5-25 

17  oo 

Sulphur  

O.Q7 

l.ot 

o.6s 

o.8s 

o.s^ 

o  46 

Phosphorus  .  .  . 
Ash  

4.58 
O.6O 

5-07 
0.78 

5.32 
1.72 

4.88 
1.72 

5-27 

17.42 

5.63 

I   17 

P2O5  in  ash.  .  .  . 
Bases  in  ash.  .  . 

O.29 
0.31 

0-55 
0.23 

1.24 
0.48 

0.94 
0.78 

10.56 
6.86 

0.69 
0.48 

These  analyses  of  nuclein  were  calculated  free  from  the  bases  of  the  ash 
and  from  nucleic  acid,  in  the  way  previously  described,  with  the  results  given 
.,1ft  table  12..^ 


TABLE  12. — Composition  of  protein  matter  contained  in  the  nuclein. 


52- 

53- 

54. 

55- 

56. 

57- 

Carbon  

P.ct. 
s?.  6s 

P.ct. 

S4.  77 

P.ct. 
SI  80 

P.ct. 
S2.^6 

P.ct. 
C2  10 

P.ct. 

si  64. 

Hydrogen  
Nitrogen  

7-23 
16.68 

7.46 
17.  "?6 

6.85 
16  •*! 

6.73 

16  •*! 

6.91 
IQ  ^1 

6.60 

18  q-i 

Sulphur  

i.  08 

2.  VI 

1.61 

i  80 

I  S^ 

I  2S 

Oxvfifen..  , 

2O  4.6 

17  84 

2T.  AT. 

22  71 

IQ  Q5 

21  58 

IOO.OO 

IOO.OO 

IOO.OO 

IOO.OO 

IOO.OO 

IOO.OO 

The  composition  of  the  protein  matter  in  54  and  55  is  very  nearly  that  of 
leucosin,  except  as  regards  sulphur,  the  amount  of  which  is  decidedly  greater. 
On  the  other  hand,  52  and  53,  which  also  were  derived  from  preparations 
whose  protein  matter  was  leucosin,  differ  in  composition  very  decidedly  from 
that  substance.  This  is  probably  because  on  pepsin  digestion  the  substance 
of  preparations  54  and  55  remained  throughout  undissolved,  whereas  52  and 
53  separated  on  pepsin  digestion  from  nearly  clear  solutions,  and  therefore 
doubtless  their  protein  matter  had  been  to  some  degree  altered  by  the  pepsin 
before  separating  as  an  insoluble  compound  with  nucleic  acid.  The  two 
nucleins,  56  and  57,  from  the  globulin,  which  also  had  separated  from 
solution,  show  similar  differences  in  composition  when  compared  with  the 
unaltered  globulin,  carbon  and  nitrogen  being  higher  and  sulphur  very  much 
higher  than  in  the  globulin. 


EXPERIMENTAL. 


53 


PROTEIN  SOLUBLE   IN   DILUTE   ALCOHOL — GLIADIN. 

As  already  stated,  wheat  flour  yields  to  dilute  alcohol  a  considerable 
amount  of  protein  matter.  Treatment  of  the  residue  remaining  after  ex- 
tracting the  flour  with  10  per  cent  sodium-chloride  brine  likewise  removes  a 
large  amount  of  protein,  as  does  also  extraction  of  the  gluten  obtained  by 
washing  the  dough  with  water.  Extracts  were  made  with  alcohol  under  all 
these  conditions,  and  the  protein  extracted  subjected  to  repeated  fractional 
precipitations. 

DIRECT  EXTRACTION  WITH  DILUTE  ALCOHOI,. 

5000  grams  of  the  straight  flour  were  extracted  with  10  liters  of  alcohol, 
0.90  sp.  gr.,  and  allowed  to  soak  over  night.  The  next  morning  the  mixture 
was  stirred,  and,  after  settling,  the  clear  solution  poured  off.  Three  liters 
more  of  alcohol,  0.90  sp.  gr.,  were  then  added,  and  after  standing  some  time 
the  supernatant  liquid  was  decanted  and  the  residue  squeezed  nearly  dry. 
The  solution  so  obtained  was  designated  ' '  extract  i . "  The  residue  was 
again  treated  with  4  liters  of  0.90  sp.  gr.  alcohol  and  pressed  nearly  dry. 
This  formed  extract  2.  The  same  process  twice  repeated  gave  two  extracts 
which,  when  united,  formed  extract  3.  Each  of  these  three  extracts,  after 
filtering  perfectly  clear,  was  separately  concentrated  to  one-third  its  volume, 
and,  after  cooling,  decanted  from  the  very  glutinous,  viscid  mass  which  had 
separated,  i  and  3  yielded  much  more  substance  than  2.  On  stirring  with 
a  glass  rod  the  precipitated  mass  formed  a  very  thick,  viscid  liquid.  This 
substance  was  in  each  case  dissolved  in  a  small  amount  of  hot  alcohol  of  0.90 
sp.  gr.,  in  which  it  was  very  soluble,  and  the  solution  was  allowed  to  cool 
over  night.  Most  of  the  substance  separated  on  cooling  and  the  liquid  was 
decanted  from  it.  The  solution  decanted  from  the  second  and  third  extracts 
was  treated  with  a  quantity  of  distilled  water  and  a  little  sodium  chloride 
added.  This  threw  down  a  small  precipitate,  which  on  standing  collected 
on  the  bottom  of  the  vessel  as  a  clear  semi-fluid  mass.  This  was  treated 
with  water,  absolute  alcohol,  and  ether,  and  yielded  7.27  grams  of  prepara- 
tion 58  from  extract  2,  and  10.7  grams  of  the  preparation  59  from  extract  3. 
These  had  the  following  compositions  : 

Preparations  58  and  59. 


Preparation  58. 

Preparation  59. 

I. 

Ash-free. 

I. 

Ash-free. 

Nitrogen  

P.  ct. 
17-05 
0.76 

P.  ct. 

17.18 

P.  ct. 

17-15 
0.65 

P.  ct. 
17.26 

Ash  

54 


THE;  PROTEINS  OE  THE  WHEAT  KERNEL. 


On  examination  both  were  found  to  contain  some  fat  which  could  not  be 
wholly  removed,  as  the  substance  had  dried  in  a  dense,  horny  form.  The 
residues  which  had  separated  from  the  solutions  just  described  were  next 
washed  by  thoroughly  intermixing  with  distilled  water.  The  water  was 
found  to  dissolve  some  of  the  protein,  which  was  subsequently  precipitated 
by  the  addition  of  a  little  sodium-chloride  solution.  After  standing  over 
night  this  precipitate  settled  to  the  bottom  of  the  vessel  in  a  transparent 
layer,  from  which  the  solution  could  be  completely  decanted.  After  treating 
this  substance  with  absolute  alcohol,  it  formed  a  voluminous  white,  porous 
mass,  which  was  digested  for  some  time  with  ether.  From  extract  i 
preparation  60  was  obtained,  weighing  12.4  grams  ;  from  extract  2,  united 
with  that  from  3,  preparation  61,  weighing  8.6  grams. 

These  preparations  were  found  on  analysis  to  have  the  following  compo- 
sitions : 

Preparations  60  and  61. 


Preparation  60. 

Preparation  61. 

I. 

II. 

Average. 

Ash-free. 

I. 

Ash-free. 

Carbon  
Hydrogen  .  . 
Nitrogen  .  .  . 
Sulphur  
Oxygen  .  . 

P.  ct. 
52.40 
6.77 
17-52 
1.05 

P.ct. 

52.58 
6.78 

17-73 
I.II 

P.ct. 

52.49 
6.78 
17.63 
1.08 

P.ct. 

52.52 
6.78 
17.64 
1.08 
21.98 

P.ct. 
52.69 
6.77 
17-74 
1.26 

P.  ct. 

52.77 
6.78 
17.77 
1.26 
21.42 

Ash  

0.06 





0.15 

IOO.OO 

IOO.OO 

The  residues  remaining  after  washing  with  distilled  water  were  then 
digested  with  alcohol  of  0.820  sp.  gr.,  which  dissolved  much  of  the 
substance.  After  standing  some  time,  the  strong  alcoholic  solutions  were 
decanted  from  the  residues  and  found  to  consist  of  milky  liquids.  The 
addition  of  a  few  drops  of  10  per  cent  sodium-chloride  solution  immediately 
produced  in  each  a  very  large,  curdy  precipitate,  the  liquids  from  which 
they  separated  being  left  perfectly  clear.  From  the  solution  from  the  first 
extract,  which  was  about  one  liter  in  volume,  32.26  grams  of  substance 
were  obtained  after  dehydration  with  absolute  alcohol  and  digestion  with 
ether.  This  was  marked  preparation  62.  From  the  second  extract  was 
similarly  obtained  preparation  63,  weighing  5.34  grams,  and  from  the  third 
extract  preparation  64,  weighing  17.43  grams.  The  filtrates  from  62  and 
63  were  found  to  be  almost  wholly  free  from  protein,  but  that  from  64  left 
on  evaporation  a  residue  which,  when  freed  from  fat,  weighed  7.53  grams, 
preparation  65. 


EXPERI  MENTAL. 

These  preparations  were  found  to  have  the  following  compositions  : 

Preparations  62  and  63. 


55 


Preparation  62. 

Preparation  63. 

I. 

Ash-free. 

I. 

II. 

Average. 

Ash-free. 

Carbon  

P.ct. 

52.59 
6.70 
17.64 

1.22 

P.ct. 
52.67 
6.71 
17.66 

1.22 

21-74 

P.  ct. 
52.28 
6.87 
17.90 
1.  21 

P.ct. 
52-54 
6.79 

P.ct. 
52.41 
6.83 
17.90 

1.  21 

P.ct. 

52-55 
6.85 
17.94 

1.  21 

21-45 

Hydrogen  .  . 
Nitrogen  .  .  . 
Sulphur  
Oxygen  .  . 

Ash  

O.I5 

O.27 

IOO.OO 

IOO.OO 

Preparations  64  and  65. 


Preparation  64. 

Preparation  65. 

I. 

II. 

Average. 

Ash-free. 

I. 

II. 

Average. 

Ash-free. 

Carbon  
Hydrogen  .  . 
Nitrogen  .  .  . 
Sulphur  .... 

P.  ct. 

P.ct. 

P.  ct. 

P.  ct. 

P.  ct. 

P.  Ct. 

P.  ct. 

P.ct. 
52.39 
6-93 
I7-3I 
1-38 
21.99 

52.52 
6.72 
17.60 

1.2^ 

52.82 
6.79 

52.67 
6.76 
17.60 
1.23 

52.74 
6.77 
17.62 
1.23 
21.64 

52.13 
6.97 

17-35 
i-35 

52.42 
6.85 
17.19 
1.41 

52.28 
6.91 
17.27 
1-38 

Oxvjjen 

Ash  

O.I4 

O.22 

IOO.OO 

IOO.OO 

The  residues  which  remained  after  treatment  with  alcohol  of  o.82osp.  gr. 
were  then  dehydrated  with  absolute  alcohol  and  digested  with  ether.  From 
extract  i  preparation  66  was  obtained,  weighing  63  grams ;  from  extract 
2  preparation  67,  weighing  2.1  grams,  and  from  extract  3  preparation  68, 
weighing  41.2  grams. 

Preparation  66,  which  constituted  the  principal  fraction  of  the  protein 
extracted,  was  further  treated  in  the  following  manner :  20  grams  were 
dissolved  in  250  cc.  of  0.90  sp.  gr.  alcohol  and  found  to  yield  a  clear  solu- 
tion, which  was  then  poured  into  800  cc.  of  absolute  alcohol,  whereby  a 
considerable  precipitate  was  at  once  separated,  leaving  the  solution  milky. 
This  substance  was  dehydrated  with  absolute  alcohol  and  digested  with 
ether,  yielding  preparation  69.  The  filtrate  was  then  treated  with  a  few 
drops  of  10  per  cent  sodium-chloride  solution,  causing  a  heavy  precipitate, 
which  on  stirring  rapidly  agglutinated  and  adhered  as  a  mass  to  the  stirring- 


THE    PROTEINS    OF   THE    WHEAT    KERNEL. 


rod.  This  was  removed,  treated  in  the  usual  manner,  and  marked  prepara- 
tion 70.  The  mother-liquor  from  which  this  separated,  after  standing  over 
night,  deposited  a  further  small  amount  of  protein,  which,  after  treatment 
with  absolute  alcohol  and  ether,  gave  preparation  7 1 . 

These  six  substances  were  analyzed  and  found  to  have  the  following 
compositions  : 

Preparations  66  to  //. 


Preparation  66. 

Preparation  67. 

I. 

Ash-free. 

I. 

II. 

\verage. 

Ash-free. 

Carbon 

P.  ct. 
52.81 
6.81 
17.66 
I.  ii 

P.ct. 
52.84 
6.81 
17.67 
I.  ii 
21-57 

P.ct. 

P.ct. 

P.ct. 

P.  ct. 

Hydrogen  .  .  . 
Nitrogen  .... 
Sulphur 

I5-42 

15.28 

15-35 

15-42 

Oxveren  .  . 

Ash  

0.06 

0.42 

0.42 

100.00 

Preparation  68. 

Preparation  69. 

I. 

II. 

Average. 

Ash-free. 

I. 

Ash-free. 

Carbon 

P.  ct. 
52.90 
6.74 
17.24 

I  O5 

P.ct. 

52.99 
6.82 
17.36 

P.ct. 

52-95 
6.78 
17.30 
1.05 

P.ct. 
53-02 
6.79 
I7-32 
1.05 
21.82 

P.ct. 

P.  ct. 

Hydrogen  .  .  . 
Nitrogen  
Sulphur 

17.67 

17.69 

OxvEren  . 

Ash  

0.15 

o.  10 

100.00 

Preparation  70. 

Preparation  71. 

I. 

II. 

Average. 

Ash-free. 

I. 

Ash-free. 

Carbon  
Hydrogen.  .  . 
Nitrogen   .  . 
Sulphur  
Oxygen  
Ash  

P.ct. 
52.15 
6-93 
I7-52 

1.. 

P.ct. 

52.35 
6.87 
17.84 

P.ct. 
52.25 
6.90 
17.68 

P.ct. 

52.33 
6.91 
17.70 

23.06 

P.ct. 
52.28 
7.12 
17.79 

P.ct. 
52.38 

7-13 
17.82 

22.67 

I 

0.15 

0.19 

ICO.OO 

IOO.OO 

EXPERIMENTAL. 


57 


If  the  preceding  analyses  are  brought  together  as  in  table  13,  the  effect 
of  the  various  fractional  solutions  and  precipitations  may  be  seen  at  a  glance. 

TABI,E  13. — Gliadin  extracted  by  direct  treatment  of  the  flour  with  alcohol. 


From  0.9  sp.  gr.  al- 
coholic solution. 

From  water  wash- 
ings. 

From  0.82  sp.  gr.  alcoholic 
solution. 

58. 

59. 

60. 

61. 

62. 

63. 

64. 

Carbon  

P.  ct. 

P.  ct. 

P.  ct. 
52.52 
6.78 
17.64 
1.08 
21.98 

P.ct. 
52.77 
6.78 
17.77 
1.26 
21.42 

P.ct. 
52.67 
6.70 
17.66 
1.22 
21-75 

P.ct. 

52.55 
6.85 

17.94 

1.  21 

21-45 

P.  ct. 

52.74 
6.77 

17.62 
1.23 

21.64 

Hydrogen    .  . 

Nitrogen  

I7.l8 

17.26 

Sulphur  

Oxveren  .  . 

Weight  of  sub- 
stance in  grams. 

IOO.OO 

12.40 

IOO.OO 

8.60 

IOO.OO 

32.26 

IOO.OO 

5.34 

IOO.OO 

17.43 

7.27 

10.70 

From 
filtrate 
from  64. 

Residue  after  extraction  with 
0.820  sp.  gr.  alcohol. 

Fractional  reprecipitations 
of  preparation  66. 

65. 

66. 

67. 

68. 

69. 

70. 

71- 

Carbon  

P.  ct. 
52.39 
S-93 
17-31 
1.38 
21.99 

P.  ct. 

52.82 
6.81 
17.67 
i.  ii 

21.57 

P.ct. 

P.ct. 

"U.O2 

P.ct. 

P.ct. 

52.33 

6.91 

17.70 
1  23.06 

P.  ct. 

52.38 
7.13 
17.82 

22.67 

Hydrogen  

6.  79 

Nitrogen  

15.42 

I7.32 
I.  OS 

17.69 

Sulphur  

Oxvsren  . 

21.82 

Weight  of  sub- 
stance in  grams. 

IOO.OO 

7.53 

IOO.OO 

63.0 

2.IO 

IOO.OO 

41.20 

IOO.OO 

IOO.OO 

It  is  evident  that  58  and  59  contain  less  nitrogen  than  the  great  bulk  of 
the  protein  extracted.  This  is  due  to  fat  which  they  were  found  to  contain 
and  which  could  not  be  wholly  removed  by  extraction  with  ether,  since  in 
drying  these  preparations  were  converted  into  the  horny  condition  which 
rendered  penetration  with  ether  impossible. 

Preparation  67  was  evidently  impure,  as  might  be  expected,  since  it  con- 
tained all  of  the  insoluble  particles  of  the  entire  extract  which  had  escaped 
filtration,  and  owing  to  its  small  amount  these  impurities  produced  a  marked 
effect  on  its  percentage  composition.  The  same  is  true  of  68,  but  as  the 
quantity  of  this  preparation  is  so  much  greater  this  contamination  has  pro- 
duced much  less  effect  on  its  composition.  Preparation  65  was  obtained  by 
evaporating  the  mother-liquor  from  64  nearly  to  dryness  and  then  extracting 
with  absolute  alcohol  and  ether.  It  would  hardly  be  expected  that  under 


THE)    PROTEINS    OF   THE    WHEAT    KERNEL. 


such  circumstances  it  would  be  entirely  pure.  The  analyses  of  the  other 
preparations  are  in  good  agreement,  and  it  is  evident  that  no  fractional 
separation  of  the  extracted  protein  is  indicated.  The  preparations  obtained 
from  solution  in  pure  water  have  the  same  composition  as  those  from  solu- 
tions in  alcohol  of  0.820  sp.  gr.,  and  also  the  same  composition  as  the 
residue  remaining  after  treatment  with  each  of  these  reagents. 

The  total  amount  of  protein  contained  in  these  several  preparations  is 
207.83  grams,  being  equal  to  4.16  per  cent  of  the  flour. 

TABLE  14. — Gliadin  extracted  from  flour  with  dilute  alcohol  after  extraction  with 
10  per  cent  sodium-chloride  solution. 


Preparation  72. 

Preparation  73. 

I. 

II. 

Average. 

Ash-free. 

I. 

II. 

Average. 

Ash-free. 

Carbon  

P.  ct. 

52.61 

P.ct. 

P.ct. 
52.61 
6.82 
17.70 

1.02 

P.ct. 
52.69 
6.84 
17-73 

1.02 
21.72 

P.tt. 
5*.65 
6.85 
17-87 
0-95 

P.ct. 

P.ct. 
52.65 
6.85 
17.87 
0-95 

P.  ct. 
52.72 
6.86 
17.89 

o-95 
21.58 

Hydrogen  .  . 
Nitrogen  
Sulphur  .... 
Oxygen  .  . 

6.82 
17.62 

1.  00 

17.78' 

1.04 

0.94 

Ash  

0.16 

o  \\ 





IOO.OO 





IOO.OO 

Preparation  74. 

Preparation  75. 

Preparation  76. 

I. 

II. 

Average. 

Ash-free. 

I. 

Ash-free. 

I. 

Ash-free. 

Carbon  
Hydrogen  .  . 
'    Nitrogen.... 
Sulphur  .... 

P.  ct. 

52.52 
6.82 
17.72 

I.IO 

P.ct. 

52.67 
6.76 

P.ct. 
52.60 
6.79 
17.72 

I.IO 

P.  ct. 
52.71 
6.81 

17-75 

I.IO 
21  6l 

P.ct. 

P.ct. 

P.  ct. 
52.62 
6.83 
17.78 
i.  08 

P.ct. 

52.65 
6.83 
17.79 
1.08 
21.65 

16.93 

17.08 

Oxygen  

Ash  

0.21 





0.91 



0.05 

IOO.OO 

IOO.OO 

EXTRACTION  WITH  DILUTE  ALCOHOL  AFTER  EXTRACTING  THE  FLOUR 
WITH  10  PER  CENT  SODIUM- CHLORIDE  SOLUTION. 

Four  kilograms  of  "straight  flour"  were  extracted  with  10  per  cent 
sodium-chloride  solution  as  long  as  anything  was  removed.  After  squeez- 
ing as  dry  as  possible  in  a  screw-press,  the  residue  was  treated  with  alcohol 
so  as  to  yield  with  the  water  retained  by  the  meal  as  nearly  as  possible  a 


EXPERIMENTAL.  59 

solution  containing  75  per  cent  of  alcohol.  After  digesting  2  days  with  the 
solvent,  the  extract  was  squeezed  out  in  a  press  and  the  process  repeated 
three  times.  Four  extracts  were  thus  obtained.  These  were  each  concen- 
trated to  small  volume,  cooled,  and  the  solution  decanted  from  the  precipi- 
tated mass.  This  was  then  washed  with  distilled  water.  After  removing 
the  salts,  the  substance  from  extracts  i  and  2  dissolved  to  some  extent  ; 
that  from  extract  3  dissolved  completely  to  a  turbid  solution.  By  adding 
sodium  chloride  the  dissolved  protein  was  precipitated. 

The  residues  remaining  after  washing  with  water  were  treated  with  abso- 
lute alcohol  and  digested  with  ether.  The  precipitates  obtained  from  the 
water  washings  by  adding  salt  were  treated  in  the  same  way.  From  extract 
i  preparation  72  was  obtained,  weighing  82  grams  ;  extract  2,  preparation 
73,  weighing  57  grams ;  from  extract  3,  after  dissolving  in  water  and 
precipitating  with  sodium  chloride,  preparation  74,  weighing  11.3  grams; 
from  extract  4  preparation  75,  weighing  only  1.35  grams,  and  from  the 
united  water  washings  of  72  and  73  preparation  76,  weighing  5.8  grams. 
The  total  weight  of  these  preparations  was  157.45  grams,  equal  to  3.94  per 
cent  of  the  flour  taken.  Their  composition  is  shown  by  the  analyses  given 
in  table  14  on  the  preceding  page. 

EXTRACTION  OF  GLUTEN  WITH  DILUTE  ALCOHOL. 

Two  kilograms  of  ' '  straight  spring- wheat  flour ' '  were  made  into  a  dough 
with  distilled  water  of  20°,  and  then  washed  in  a  stream  of  river  water  of 
5°.  After  washing  until  nearly  all  the  starch  was  removed,  the  gluten  was 
chopped  up  fine  and  digested  with  alcohol  of  0.90  sp.  gr.  at  a  tempera- 
ture of  about  20°.  This  extraction  was  continued  with  repeatedly  re- 
newed portions  of  alcohol  of  the  same  strength  as  long  as  anything  was 
removed.  The  extracts  were  united,  filtered  perfectly  clear,  and  concen- 
trated to  about  one-fourth  their  original  volume.  The  residual  solution 
was  then  cooled  and  allowed  to  stand  over  night  to  deposit  the  separated 
gliadin.  The  supernatant  solution  was  poured  off  and  the  large  amount 
of  protein  which  had  separated  was  then  dehydrated  by  treatment  with 
absolute  alcohol.  The  decanted  mother-liquor  from  which  this  protein  had 
separated,  and  also  the  strong  alcoholic  solution  which  resulted  from  dehy- 
drating the  precipitated  mass,  were  each  precipitated  by  adding  a  little 
sodium-chloride  solution.  The  three  products  thus  obtained  were  united, 
digested  with  fresh  quantities  of  absolute  alcohol  in  order  to  complete  the 
dehydration,  and  then  extracted  with  absolute  ether.  Dried  over  sulphuric 
acid,  the  preparation  weighed  82  grams,  and  formed,  therefore,  4. 10  per  cent 
of  the  flour  taken.  Dried  at  110°,  this  substance  had  the  composition 
shown  under  the  head  "  Preparation  77  "  in  the  table  on  page  60. 


6o 


THE    PROTEINS    OF   THE    WHEAT    KERNEL. 


Thirty  grams  of  preparation  77  were  then  dissolved  in  alcohol  of  0.90  sp.  gr. , 
and  the  clear  solution  evaporated  to  small  volume,  cooled,  and,  as  no  protein 
separated,  strong  alcohol  was  added  until  a  considerable  precipitate  resulted, 
equal  to  about  one-half  the  dissolved  protein.  This  precipitate,  which,  if 
the  extracted  protein  were  a  mixture,  as  stated  by  Ritthausen,  would  con- 
tain the  bulk  of  the  substances  insoluble  in  strong  alcohol,  weighed  12  grams. 
This  was  marked  preparation  78. 

The  solution  from  which  this  substance  had  separated  must  have  con- 
tained the  chief  part  of  the  protein  called  by  Ritthausen  gluten-fibrin.  It 
was  then  concentrated  to  small  volume,  cooled,  water  added  until  a  con- 
siderable precipitate  resulted,  the  solution  then  heated  until  all  dissolved, 
and,  after  cooling,  the  mother-liquor  was  decanted  from  the  separated 
protein.  This  process  was  repeated  four  times,  and  the  precipitate  finally 
obtained  dehydrated  with  absolute  alcohol  and  digested  with  ether.  This 
preparation,  79,  weighed  1.6  grams. 

Preparations  77,  78,  and  79. 


Preparation  77. 

Preparation  78. 

Preparation  79. 

I. 

Ash-free. 

I. 

Ash-free. 

I. 

Ash-free. 

Carbon  
Hydrogen  .  . 
Nitrogen  .  .  . 
Sulphur  

P.  ct. 

52.33 
6.63 

17-57 
1.08 

P.ct. 
52.58 
6.67 

17-65 
1.08 

22.02 

P.ct. 
52.82 
6.77 
17.62 
1.09 

P.ct. 

52.68 
6.78 

17.65 
1.09 
21.  80 

P.ct. 

52.82 
7.18 
17-57 

P.ct. 

52.84 
7.I8 
17-57 
22.41 

Ash  

0.50 

0.19 

0.04 

100.00 

IOO.OO 

IOO.OO 

It  is  clear  from  these  analyses  that  no  separation  into  proteins  of  differing 
composition  had  thus  been  effected. 

EXTRACTION  OF  "SHORTS"  WITH  DILUTE  ALCOHOI,. 

Two  kilograms  of  ' '  shorts ' '  from  the  spring- wheat  flour  were  extracted 
with  alcohol  of  0.90  sp.  gr.  and  the  extract  squeezed  out  with  a  screw- press. 
The  extract,  which  was  a  deep  red-brown  in  color,  was  filtered  perfectly 
clear,  and  then  concentrated  by  distillation  to  about  one-third.  On  cooling, 
the  protein  separated,  leaving  the  mother-liquor  as  a  deep  coffee-brown 
liquid.  This  was  decanted,  the  precipitate  dissolved  in  alcohol  of  0.90 
sp.  gr. ,  and  again  precipitated  by  concentration  and  cooling ;  the  strongly 


EXPERIMENTAL. 


61 


colored  mother-liquor  was  decanted  and  this  process  repeated.  The  pre- 
cipitated protein  was  then  again  dissolved  in  a  little  dilute  alcohol,  and  the 
resulting  solution  poured  into  absolute  alcohol,  thereby  precipitating  the 
greater  part  of  the  protein  and  leaving  the  alcohol  strongly  colored.  The 
precipitate  was  thus  freed  from  a  very  considerable  part  of  its  coloring 
matter.  After  digestion  with  absolute  alcohol,  and  finally  with  ether,  this 
preparation,  80,  was  dried  and  analyzed  with  the  result  shown  in  the 
table  below : 

Owing  to  the  fact  that  this  preparation,  80,  was  still  contaminated  with 
coloring  matter,  and  also  showed  slight  differences  in  composition  from  the 
protein  extracted  by  similar  treatment  from  the  flour,  it  was  subjected  to 
further  treatment  with  a  view  to  its  more  complete  purification. 

Apart  of  the  preparation  was  dissolved  in  isocc.  of  alcohol  of  0.90  sp.  gr. 
and  the  solution  poured  into  1000  cc.  of  absolute  alcohol.  This  produced  a 
turbid  liquid,  which,  on  adding  a  drop  or  two  of  sodium-chloride  solution, 
gave  a  heavy  precipitate  that  rapidly  settled,  leaving  the  alcohol  colored 
yellow.  This  precipitate  was  again  dissolved  in  diluted  alcohol,  and  pre- 
cipitated by  pouring  into  ether  in  order  to  remove  anything  soluble  in  this 
liquid.  No  coloring  matter  was  thus  removed.  The  precipitate  was  then 
digested  with  absolute  alcohol,  yielding  preparation  81. 

The  strong  alcoholic  solution  from  which  this  preparation  had  separated 
on  longer  standing  deposited  a  small  amount  of  substance  which,  when 
dehydrated,  yielded  preparation  82.  Analysis  showed  these  two  substances 
to  have  the  composition  shown  in  the  following  table  : 

Preparations  80,  81,  and  82. 


Preparation  80. 

Preparation  81. 

Preparation  82. 

I. 

Ash- 
free. 

I. 

II. 

Aver- 
age. 

Ash- 
free. 

I. 

II. 

Aver- 
age. 

Ash- 
free. 

Carbon.  .  .  . 
Hydrogen  . 
Nitrogen  .  . 
Sulphur.  .  . 

P.  ct. 

52.75 
6.96 
17.22 
1.36 

P.  ct. 
53-25 
7.02 

17-38 
1-37 
20.98 

P.  ct. 
52.57 
6.74 

17-39 

P.ct. 
52.52 
6.80 

P.ct. 
52.55 
6.77 
17-39 

P.ct. 
52-85 
6.81 
17.48 

22.86 

P.ct. 

52.43 
6.79 

17.59 

P.ct. 

52.56 

6.88 

P.ct. 
52.50 
6.84 

17-59 

P.ct. 
52.74 
6.87 
17-67 

22.72 

Oxygen  . 
Ash  

o-95 

/"" 
o.54 



0.46 





IOO.OO 



IOO.OO 



IOO.OO 

These  figures  show  that  the  protein  extracted  from  the  "  shorts  "  has  the 
same  composition  as  that  similarly  obtained  from  the  flour. 


02  THE    PROTEINS    OF   THE    WHEAT    KERNEL. 

PROTEIN  EXTRACTED  BY  ALCOHOL  FROM  WHOLE-WHEAT  FLOUR. 

As  Ritthausen  and  probably  others  used  whole-wheat  flour  in  extracting 
the  various  proteins  described  by  them  as  soluble  in  dilute  alcohol,  it  was 
thought  best  to  carry  out  some  extractions  with  meal  obtained  by  grinding 
the  entire  wheat  kernel  in  the  laboratory. 

Preparations  83-88. 


Preparation  83. 

Preparation  84. 

I. 

II. 

Aver- 
age. 

Ash- 
free. 

I.            II. 

Aver- 
age. 

Ash- 
free. 

Carbon  

P.  ct. 

52.88 
7.00 
17-47 

I.  AT. 

P.  Ct. 
52.68 
6.97 
17.48 

P.  ct. 

52.78 
6.98 
17.48 
1-43 

P.  ct. 
52.90 
6.99 
I7-52 
1-43 
21.  16 

P. 
52. 
6. 

17- 
o. 

:t.        P.  ct. 

59      52.73 
37        6.80 
32      18.06 

92     . 

P.ct. 
52.66 
6.84 
17.99 
0.92 

P.ct. 
52.89 
6.87 
1  8.  06 
0.92 
21.26 

Hydrogen... 
Nitrogen  .  .  . 
Sulphur.  .  .  . 

Oxygen  .  . 

Ash  

O  2^ 

O.d5 

IOO.OO 

100.00 

Preparation  85. 

Preparation  86. 

I. 

II. 

Average. 

Ash-free. 

I. 

Ash-free. 

Carbon  .... 

P.  Ct. 

51-03 
6-59 
16.98 
0.92 

P.  ct. 

50.93 
6.50 
17.08 

P.ct. 
50.98 

6-55 
17.03 
0.92 

P.ct. 

53-i6 
6.83 

17-75 
0.96 
21.30 

P.ct. 

52.64 

6.86 

17-49 

1 

P.ct. 

52.82 
6.88 
17-55 
22.75 

Hydrogen  

Nitrogen  

Sulphur  

Oxygen  .  . 

1  
0-35 

Ash  

4.11 

4-03 

4.07 

IOO.OO 

IOO.OO 

Preparation  87. 

Preparation  88. 

I. 

Ash-free. 

I. 

II. 

Average. 

Ash-free. 

Carbon  

P.  ct. 

52.66 
6.80 
17.62 

}  

0.04 

P.ct. 

52.68 
6.81 
17-63 

22.88 

P.  ct. 

52.03 
6.66 
17.48 
f    1.08 

P.ct. 

51.88 
6.69 

P.ct. 

51-96 
6.68 
17.48 
i.  08 

P.ct. 

52-24 
6.71 
17-57 
1.08 
22.40 

Hydrogen  

Nitrogen  . 

Sulphur  

Oxvcren 

Ash 

0.52 

100,00 

IOO.OO 

EXPERIMENTAL.  63 

One  kilogram  of  whole  spring- wheat  flour,  freshly  ground,  was  made  into  a 
dough,  and  the  gluten  obtained  from  this  by  washing  with  water  was  then 
chopped  fine  and  thoroughly  extracted  with  alcohol  of  0.90  sp.  gr.,  the 
yellow  extract  concentrated,  and  the  protein  separated  by  cooling.  The 
deposit  thus  produced  was  dissolved  as  far  as  possible  in  dilute  alcohol,  and 
the  insoluble  substance,  which  was  coagulated  protein,  was  washed  with 
dilute  alcohol,  absolute  alcohol,  and  ether.  This  was  preparation  83. 

The  solution  filtered  from  83  was  poured  into  absolute  alcohol  and  a  small 
amount  of  protein  separated  ;  this  was  treated  with  absolute  alcohol  and 
ether  in  the  usual  way,  yielding  preparation  84.  The  filtrate  from  84  was 
concentrated  to  small  volume  and  poured  into  absolute  alcohol,  whereby 
nearly  all  the  protein  was  precipitated.  This  substance  was  dehydrated  with 
absolute  alcohol  and  digested  with  ether,  giving  preparation  85.  These  three 
bodies  had  the  composition  shown  by  the  figures  for  preparations  83,  84, 
and  85  in  the  table  on  page  62. 

In  a  similar  manner  an  extract  was  made  of  winter-wheat  meal  obtained 
by  grinding  the  entire  wheat  kernel  in  the  laboratory,  the  alcoholic  extract 
concentrated  to  about  one-third  of  its  volume,  cooled,  and  the  solution 
decanted  from  the  deposit.  This  was  then  dissolved  in  alcohol  of  0.90 
specific  gravity  and  the  coagulated  protein  filtered  off,  washed  with  dilute 
alcohol,  digested  with  absolute  alcohol  and  then  with  ether,  giving  prepara- 
tion 86. 

The  solution  filtered  from  this  preparation  was  concentrated  to  small 
volume,  cooled,  and  the  protein  separated  was  digested  with  absolute  alcohol 
and  with  ether,  yielding  preparation  87.  (See  table  on  p.  62. )  The  complete 
extraction  of  this  protein  from  the  gluten  is  very  difficult,  a  little  generally 
remaining  in  the  insoluble  residue  after  extracting  with  dilute  alcohol.  In 
one  case  the  residue  thus  remaining  was  dissolved  in  o.  2  per  cent  potassium- 
hydroxide  solution,  and  the  resulting  solution,  after  standing  some  time  to 
deposit  suspended  impurities,  was  decanted  and  precipitated  with  dilute 
hydrochloric  acid.  This  precipitate  was  washed  by  decantation  with  water 
and  then  digested  for  some  time  with  dilute  alcohol.  The  alcoholic  solution 
was  then  filtered  and  concentrated  to  small  volume  and  cooled.  The  protein 
separated  was  then  digested  with  absolute  alcohol  and  with  ether  and  dried 
at  1 10°  for  analysis. 

From  this  analysis  it  is  seen  that  the  protein  soluble  in  dilute  alcohol  is 
not  changed  in  composition  by  solution  with  potassium  hydroxide,  nor  is  its 
solubility  altered,  so  far  as  could  be  learned. 

In  order  to  facilitate  a  comparison  of  these  analyses  they  have  been 
brought  together  in  table  15. 


64  THE    PROTEINS   OF   THE    WHEAT    KERNEL. 

TABLE  15. — Composition  of  protein  extracted  by  dilute  alcohol. 


Carbon. 

Hydrogen. 

Nitrogen. 

Sulphur. 

Oxygen. 

Total. 

P.ct. 

P.ct. 

P.ct. 

P.ct. 

P.ct. 

P.ct. 

'  s8* 

17.18 

591 

17.26 

60 

52.52 

6.78 

17.64 

i.  08 

21.98 

IOO 

61 

52.77 

6.78 

17-77 

1.26 

21.42 

IOO 

62 

52.67 

6.70 

17.66 

1.22 

21-75 

IOO 

By  direct  extrac- 
tion   

63 
64 

52.55 
52.74 

6.85 
6-77 

17-94 
17.62 

1.  21 
1.23 

21.45 
21.64 

IOO 
IOO 

65 

52-39 

6-93 

I7-3I 

1.38 

21.99 

IOO 

66 

52.84 

6.81 

17.67 

I.  II 

21.57 

IOO 

68 

53-02 

6.79 

17.32 

1.05 

21.82 

IOO 

69 

17.69 

^ 

70 

52.33 

6.91 

•          J 

17.70 

23.06 

IOO 

L7i 

52.38 

7.13 

17.82 

22.67 

IOO 

After  extraction 

52.69 

6.84 

17-73 

I.  O2 

21.72 

IOO 

with  10  per  cent 

^73 

52.72 

6.86 

17.89 

o-95 

21.58 

IOO 

sodium  -  chlo- 

74 

52.71 

6.81 

17-75 

1.  10 

21.63 

IOO 

ride  solution  .  . 

.76 

52.65 

6.83 

17.79 

1.08 

21.65 

IOO 

From   gluten, 

'77 

52-58 

6.67 

17.65 

1.08 

22.02 

IOO 

spring  -  wheat  - 

78 

52.68 

6.78 

17.65 

1.09 

21.80 

IOO 

flour  

7Q 

52.84 

7.18 

17.  "57 

22.  /IT 

IOO 

From   gluten, 
whole   spring-  - 
wheat  flour.  .  . 

•>  to-*  IOOQ 

CO  COCO  00 

52.90 
52.89 

52.24 

/  *  * 

6.99 
6.87 
6.83 
6.71 

/  *  \J  1 

I7-52 
18.06 
17-75 
17-57 

1-43 
0.92 
0.96 

1.08 

T~ 

21.  16 
21.26 
21.30 
22.40 

IOO 
IOO 
IOO 
IOO 

From   gluten,  }  g6 
whole   winter-  >-g 
wheat  flour  .  .  .  J    ' 

52.82 
52.68 

6.88 
6.81 

17-55 
17.63 

22.75 

22.88 

IOO 

IOO 

From     wheat 
"shorts"  

'80 

53-25 
52.85 

7.02 
6.81 

17-38 
17.48 

1-37 

22 

20.98 

86 

IOO 
IOO 

.82 

52.74 

6.87 

17.67 

22.72 

IOO 

Average  of  preceding 

figures  

52.72 

6.86 

17.66 

I.I4 

21.62 

IOO 

1  Omitted  in  average. 

THE  PROPORTION   OF   GLUTAMINIC  ACID   YIELDED   BY  VARIOUS   FRACTIONS 
OF   THE   ALCOHOL-SOLUBLE   PROTEIN. 

The  results  of  this  extensive  fractionation  of  the  alcohol-soluble  protein 
give  no  evidence,  based  on  the  ultimate  composition  of  the  many  fractions, 
of  the  presence  of  more  than  one  protein  substance.  This,  however,  does 
not  justify  the  conclusion  that  only  one  such  substance  exists.  In  view  of 
the  recent  positive  statements  of  Kossel  &  Kutscher  and  of  Konig  &  Rintelen 
it  is  important  to  obtain,  if  possible,  further  evidence  based  on  the  propor- 
tion of  some  of  the  decomposition  products  yielded  by  the  various  fractions 
of  this  protein.  As  gliadin  yields  a  relatively  large  amount  of  glutaminic 


EXPERIMENTAL.  65 

acid  when  decomposed  by  boiling  with  strong  hydrochloric  acid,  determina- 
tions of  the  quantity  of  glutaminic  acid  obtained  from  various  fractions 
have  been  made. 

The  first  difficulty  encountered  in  examining  the  evidence  that  has  been 
offered  respecting  the  existence  of  several  alcohol-soluble  proteins  in  wheat 
flour  lay  in  the  impossibility  of  following  Ritthausen's  directions  for  pre- 
paring these,  since  many  evidently  important  details  are  omitted  in  the 
description  of  his  methods. 

Kossel  &  Kutscher  state  that  their  products  were  made  according  to  Ritt- 
hausen's directions,  but  give  no  details,  nor  do  they  state  which  method 
they  employed.  Kutscher  concludes  another  paper  by  the  statement  that 
' '  the  wheat  gluten  consists  of  gluten-casein  wholly  insoluble  in  cold  60  per 
cent  alcohol ;  gluten-fibrin,  but  little  soluble,  and  gliadin,  easily  soluble  in 
cold  60  per  cent  alcohol." 

Although  the  writer  has  made  a  very  large  number  of  preparations  repre- 
senting fractions  of  this  protein  substance  dissolved  by  alcohol  of  various 
degrees  of  strength,  he  has  never  obtained  any  that  were  not  either  com- 
pletely soluble  in  cold  alcohol  of  60  per  cent  by  volume,  or  else  contained 
such  insignificant  quantities  which  did  not  dissolve  that  he  has  found  it 
impossible  to  make  from  them  a  preparation  of  ' '  gluten-fibrin ' '  suitable 
for  further  examination.  He  has,  therefore,  been  unable  to  repeat  the 
work  of  Kossel  &  Kutscher  and  is  entirely  at  a  loss  to  understand  how  their 
preparation  of  ' '  gluten-fibrin ' '  was  obtained. 

Konig  &  Rintelen  describe  their  procedure  in  more  detail.  These  inves- 
tigators extracted  wheat  gluten  with  absolute  alcohol,  added  ether  to  the 
alcoholic  extract,  and  united  the  precipitate  produced  with  the  extracted 
gluten.  This  latter  was  then  extracted  with  65  per  cent  alcohol,  and  to  the 
extract  alcohol  was  added  until  the  mixture  contained  88  to  90  per  cent  of 
alcohol. 

After  decanting  from  the  precipitate  that  had  formed,  the  solution  was 
filtered  clear  and  evaporated  to  dryness  on  the  water-bath,  finely  pulverized, 
and  extracted  with  ether  to  remove  fat.  As  all  of  the  fat  could  not  be  thus 
removed,  the  mass  was  again  dissolved  in  alcohol,  to  which  some  potassium 
hydroxide  was  added,  and  this  solution  shaken  out  several  times  with  ether. 
The  weakly  alkaline  solution  was  then  exactly  neutralized  with  hydrochloric 
acid  and  evaporated  on  the  water-bath.  The  product  thus  obtained  was 
their  "gluten-fibrin." 

The  precipitate  produced  by  88  to  90  per  cent  alcohol  was  washed  with 
alcohol  of  the  same  strength  and  dissolved  in  a  little  65  per  cent  alcohol. 
From  this  one-half  of  the  alcohol  was  distilled  off  and  the  residual  solution 
cooled,  when  a  precipitate  separated.  From  this  the  solution  was  decanted, 
leaving  a  mass  of  gliadin.  The  solutions  which  remained  from  several  such 
5 


66  THE   PROTEINS   OP   THE    WHEAT    KERNEL. 

precipitates  were  united  and  distilled  until  one-third  of  the  solvent  was 
removed.  On  cooling  the  residual  solution,  a  deposit  formed  which  they 
considered  to  be  a  mixture  of  gliadin  and  mucedin.  The  solution  decanted 
from  this  deposit  was  evaporated  to  dryness,  and  yielded  a  considerable 
residue  of  "mucedin." 

The  analyses  of  these  products  showed  that  the  gliadin  thus  prepared  had 
the  same  composition  as  that  obtained  by  the  writer,  as  well  as  that  made 
by  Ritthausen,  while  the  "gluten-fibrin"  and  mucedin  contained  about  i 
per  cent  less  nitrogen  and  much  more  carbon. 

Konig  &  Rintelen  obtained  their  mucedin  from  the  nearly  aqueous  solu- 
tions remaining  after  separating  the  gliadin  by  evaporating  to  dryness.  The 
writer  assumes  that  the  residue  which  remained  was  subjected  to  some  fur- 
ther purification,  but  concerning  this  they  say  nothing.  The  solution  con- 
tains many  impurities,  and  when  the  protein  substance  in  it  has  been 
properly  purified  it  has  the  properties  and  composition  of  gliadin. 

Although  large  quantities  of  gliadin  have  been  made  in  this  laboratory 
and  subjected  to  very  careful  and  extensive  fractionation,  no  evidence  what- 
ever of  the  existence  of  "  mucedin  "  has  been  obtained. 

Furthermore,  it  would  seem  improbable  that  our  gliadin  could  be  con- 
taminated by  "gluten-fibrin"  and  "mucedin,"  which  the  writer  certainly 
did  not  succeed  in  separating  from  it,  and  at  the  same  time  show  so  close 
an  agreement  in  composition  with  that  of  Konig  &  Rintelen,  from  which 
they  suppose  that  both  of  these  proteins  had  been  carefully  removed.  It  is 
also  improbable  that  Kjeldahl  should  have  found  the  specific  rotation  uniform 
for  numerous  preparations  of  the  alcohol-soluble  protein  if  the  material 
which  he  examined  was  a  mixture  of  three  different  substances  ;  nor,  if  this 
were  the  case,  could  his  determination  of  (a)D  — 92°  be  expected  to  agree 

20° 
so  closely  with  that  made  by  Osborne  &  Harris,1  («)^ 92.3°,  and  by 

Mathewson,2  (a)  ~ 91. 95°. 

The  composition  of  both  ' '  gluten-fibrin ' '  and  ' '  mucedin  ' '  differs  from 
that  of  gliadin  just  as  one  would  expect  if  these  former  substances  were 
slightly  altered,  and  somewhat  impure  products  obtained  from  gliadin. 

Until  more  convincing  evidence  of  the  existence  of  "  gluten-fibrin  "  and 
"mucedin"  as  distinct  protein  substances  is  brought  forward,  they  can  not 
be  considered  to  be  original  constituents  of  the  wheat  kernel. 

Kutscher  has  stated  that  there  is  a  wide  difference  in  the  proportion  of 
glutaminic  acid  which  is  yielded  by  gliadin  and  gluten-fibrin.  The  writer 

1  Osborne  &  Harris,  Journal  American  Chemical  Society,  1903,  xxiv,  p.  844. 

2  Mathewson,  Journal  American  Chemical  Society,  1906,  xxxvin,  p.  1482. 


EXPERIMENTAL.  67 

has  therefore  determined  the  amount  of  this  amino-acid  which  was  yielded 
by  several  preparations  of  the  alcohol-soluble  protein,  one  of  which  repre- 
sented the  fraction  of  the  protein  soluble  in  the  strongest  alcohol,  and  should 
therefore,  according  to  Ritthausen,  consist  chiefly  of  gluten-fibrin.  The 
amount  of  glutaminic  acid  in  gliadin  was  first  determined  by  boiling  100 
grams  of  gliadin,  equal  to  93  grams  moisture  free,  for  14  hours  with  200  cc. 
of  concentrated  hydrochloric  acid.  After  standing  on  ice  for  3  days,  the 
entire  solution  solidified  to  a  thick  mass  of  crystals,  which  was  sucked  out 
with  a  pump  and  washed  with  ice-cold  alcoholic  hydrochloric  acid.  When 
dried  over  sodium  hydroxide,  this  crude  glutaminic  acid  hydrochloride 
weighed  58.33  grams.  The  filtrate  and  washings  on  concentration  gave  by 
similar  treatment  2.58  grams  more,  making  the  total  crude  glutaminic  acid 
hydrochloride  60. 01  grams.  This  product  was  then  dissolved  in  water  freed 
from  color  with  animal  charcoal  and  recrystallized.  After  removing  ammo- 
nium chloride  by  boiling  with  a  slight  excess  of  barium  hydroxide  and  the 
barium  with  an  equivalent  quantity  of  sulphuric  acid,  43.02  grams  of  pure 
glutaminic  acid  hydrochloride  were  obtained,  which  are  equal  to  34.42  grams 
of  the  free  acid,  or  37  per  cent  of  the  gliadin. 

Nitrogen:  0.5092  gram  substance,  dried  at  100°,  gave  NH3  =  3.86  HC1  (i  cc.  HC1  = 

o.oi  gram  N)  =  7.58  p.  ct.  N. 
Calculated  for  C5H10O4NC1,  7.64  p.  ct.  N. 

In  confirmation  of  these  figures  this  determination  was  repeated  with 
two  fractions  of  the  alcohol-soluble  protein  of  wheat  gluten  which  had  been 
separated  from  relatively  strong  alcoholic  solutions,  and  should  therefore 
have  contained  a  large  proportion  of  ' '  gluten-fibrin ' '  if  the  statements 
respecting  the  solubility  of  this  substance  are  correct. 

Two  portions  of  different  preparations  of  the  air-dry  substance,  equivalent 
to  18.62  and  14.65  grams  dried  at  110°,  were  hydrolyzed  as  before,  their 
solutions  saturated  with  hydrochloric  acid,  and  kept  for  some  time  on  ice. 
The  glutaminic  acid  hydrochloride  which  separated  was  filtered  out,  washed 
with  alcoholic  hydrochloric  acid,  and  freed  from  ammonia  by  evaporating 
with  an  excess  of  baryta  and  from  barium  by  an  equivalent  quantity  of 
sulphuric  acid.  The  solution  was  then  decolorized  with  animal  charcoal 
and  evaporated  with  an  excess  of  hydrochloric  acid  until  crystallization 
began.  The  glutaminic  acid  hydrochloride  which  separated,  when  washed 
with  ice-cold  alcoholic  hydrochloric  acid  and  dried,  weighed,  respectively, 
8.69  and  6.27  grams,  equivalent  to  37.33  and  34.2  per  cent  of  free  glutaminic 
acid  in  the  protein. 

Another  attempt  was  made  to  isolate  a  fraction  of  ' '  gluten-fibrin ' '  by 
dissolving  200  grams  of  a  preparation  representing  the  total  alcohol-soluble 
protein  of  wheat  gluten  in  a  mixture  of  900  cc.  of  absolute  alcohol  and 


68  THE  PROTEINS  OF  THE  WHEAT  KERNEL. 

600  cc.  of  water — that  is,  in  alcohol  of  60  per  cent  by  volume.  Although 
the  solution  was  somewhat  turbid,  nothing  was  deposited,  even  on  long 
standing.  300  cc.  of  water  were  then  added  to  the  solution,  making  the 
alcohol  50  per  cent,  but  still  nothing  separated.  The  strength  of  the  alcohol 
was  therefore  raised  to  75  per  cent  by  adding  1800  cc.  of  absolute  alcohol, 
and  a  very  large  precipitate,  A,  at  once  separated.  By  adding  2000  cc.  of 
absolute  alcohol  to  the  clear  solution  from  which  A  separated  another  large 
precipitate  (B)  was  produced,  and  in  the  filtrate  from  B  a  third  precipitate 
(C)  resulted,  when  a  further  large  quantity  of  absolute  alcohol  was  added. 
This  last  product  weighed  20  grams  and  constituted  only  10  per  cent  of  the 
total  protein.  The  solution  from  which  C  separated  contained  only  traces 
of  protein,  and  C,  therefore,  represented  the  fraction  of  the  whole  protein 
soluble  in  the  strongest  alcohol,  and  should  consequently  contain  much 
"  gluten-fibrin."  In  17.5  grams  of  this  substance  dried  at  110°  the  gluta- 
minic  acid  produced  by  decomposing  with  hydrochloric  acid  was  determined. 
By  proceeding  in  the  same  manner  as  in  the  experiment  last  described  7.8914 
grams  of  pure  glutaminic  acid  hydrochloride  were  obtained,  which  are  equiv- 
alent to  6.2131  grams  of  the  free  acid,  or  35.5  per  cent. 

Nitrogen:  0.7296  gram  substance,  dried  at  110°,  gave  NH3  =  5.66  cc.  HC1  (i  cc.  HC1 

=  o.oioo  grain  N)  =  7.75  p.  ct.  N. 
Calculated  for  C5H9NO4HC1,  7.64  p.  ct.  N. 

This  result  seems  to  furnish  conclusive  evidence  that  there  is  no  fraction, 
soluble  in  very  strong  alcohol,  to  be  obtained  from  the  alcohol-soluble  pro- 
tein of  wheat  gluten  that  is  characterized  by  yielding  a  relatively  small 
proportion  of  glutaminic  acid. 

Since  Kutscher  decomposed  his  proteins  with  sulphuric  acid,  while  hydro- 
chloric acid  was  used  in  the  preceding  experiments,  the  following  experi- 
ment was  made  in  order  to  determine  whether  the  higher  yield  obtained  by 
us  might  not  be  due  to  this  fact.  Accordingly  50  grams  of  one  of  the 
preparations  of  gliadin,  from  which  37  per  cent  of  glutaminic  acid  had  pre- 
viously been  isolated,  were  boiled  for  14  hours  with  a  mixture  of  150  grams 
of  sulphuric  acid  and  300  cc.  of  water. 

The  resulting  solution  was  treated  with  an  excess  of  baryta  and  the  ammo- 
nia expelled  by  evaporation.  The  barium  was  then  removed  by  an  equiv- 
alent amount  of  sulphuric  acid  and  the  filtered  solution  evaporated.  Some 
tyrosine  separated,  which  was  filtered  out,  and  the  evaporation  continued 
until  the  volume  was  quite  small.  On  standing,  an  abundant  quantity  of 
crystals  of  free  glutaminic  acid  separated,  and  from  the  mother-liquor,  by 
further  concentration  and  standing,  a  second  crop  of  crystals  was  obtained. 
After  recrystallizing  several  times,  8.48  grams  of  pure  glutaminic  acid  were 
obtained,  which  contained  9.43  per  cent  of  nitrogen. 


EXPERIMENTAL. 


Nitrogen;  0.6094  gram  substance,  dried  at  110°,  gave  NH3  =  5.75  cc.  HC1  (i  cc.  HC1 
=  o.oioo  gram  N)  =  9.43  p.  ct.;  calculated  for  C5H9NO4,  9.48  p.  ct. 

From  the  mother-liquors  which  yielded  this  glutaminic  acid  there  were 
further  separated,  by  saturating  with  hydrochloric  acid  and  proceeding  in 
the  manner  already  described,  4.055  grams  of  well-crystallized  hydrochloride, 
which  contained  7.73  per  cent  of  nitrogen. 

Nitrogen:  0.4983  gram  substance,  dried  at  110°,  gave  NH3  =  3.8s  cc.  HC1  (i  cc.  HC1 
=  0.0100  gram  N)  =  7.73  p.  ct.  N  ;  calculated  for  C5H9NO4HC1,  7.64  p.  ct.  N. 

The  free  glutaminic  acid  corresponding  to  this  amount  of  the  hydro- 
chloride  is  3.244  grams,  making  a  total  of  1 1.724  grams.  Since  the  50  grams 
of  air-dry  gliadin  were  equivalent  to  46.33  grams  dried  at  110°,  this  amount 
of  glutaminic  acid  is  equal  to  25.3  percent,  or  about  the  same  proportion  as 
Ritthausen  found  in  his  preparation  of  ' '  mucedin ' '  after  decomposing  with 
sulphuric  acid,  but  much  more  than  the  19.81  per  cent  found  by  Kutscher 
in  the  same  substance.  The  amount,  however,  was  only  about  two-thirds 
as  much  as  that  found  after  decomposing  with  hydrochloric  acid,  and 
although  the  separation  was  not  complete  there  was  no  reason  to  suppose 
that  more  remained  in  the  mother-liquors  in  one  case  than  in  the  other.  It 
is  possible,  however,  that  the  very  large  precipitate  of  barium  sulphate  that 
formed  on  removing  the  sulphuric  acid  retained  a  considerable  part  of  the 
glutaminic  acid  even  after  extensive  washing  with  hot  water. 

In  conclusion,  the  results  of  these  determinations  are  here  brought  together 
that  they  may  be  more  readily  compared : 

TABUS  16. — Percentage  of  glutaminic  acid  yielded  by  gliadin. 


Preparation. 

Decomposed  by  — 

HG1. 

H2SO4. 

i  

2  

*. 

P.  ct. 

37.00 

37-33 
34-20 
35-50 

P.ct. 

4  

5.  . 

25-3 

All  these  are  minimal  figures,  since  in  each  case  some  glutaminic  acid 
still  remained  in  the  mother-liquors,  but  it  does  not  seem  probable  that  more 
than  relatively  insignificant  quantities  were  thus  lost. 

From  these  results  it  would  appear  that  Kutscher 's  determinations  of 
glutaminic  acid  fall  far  short  of  the  actual  quantity  of  this  substance  yielded 
by  the  alcohol-soluble  protein  of  wheat,  and  that  they  therefore  afford  no 


7O  THE    PROTEINS   OF   THE    WHEAT    KERNEL. 

evidence  which  justifies  the  conclusion  that  this  substance  consists  of  two 
distinct  protein  bodies.  Fractional  precipitations  of  this  alcohol-soluble 
protein  yield  practically  the  same  large  proportion  of  glutaminic  acid,  so 
that,  in  view  of  their  very  close  agreement  in  composition  and  properties, 
both  physical  and  chemical,  there  is  every  reason  to  believe  that  only  one 
such  protein  is  present.  Gliadin  yields  a  remarkable  proportion  of  gluta- 
minic acid,  much  in  excess  of  that  from  any  other  known  protein  and 
greater  than  that  of  any  single  decomposition  product  yet  obtained  in  a 
pure  state  from  any  other  true  protein  substance,  the  protamines,  of  course, 
excepted. 

HYDROLYSIS   OF 


There  being  no  sufficient  evidence  that  more  than  one  alcohol-soluble 
protein  occurs  in  the  wheat  kernel,  no  attempt  has  been  made,  in  preparing 
large  quantities  of  gliadin  for  the  present  investigation,  to  subject  the  pro- 
tein matter  extracted  by  alcohol  to  any  fractional  precipitation,  but  it  was 
separated  as  completely  as  possible  from  all  other  substances  soluble  in  water, 
alcohol,  and  ether. 

The  gliadin  for  this  investigation  was  prepared  entirely  from  gluten,  as 
thereby  the  water-soluble  constituents  of  the  seed  are  more  completely  re- 
moved than  by  any  other  method  of  preparation  which  can  be  readily  used 
on  a  large  scale.  The  wheat  flour  was  kneaded  into  dough  in  a  domestic 
'  '  bread-mixer,  '  '  and  then  under  water  in  a  specially  constructed  kneading- 
machine.  After  frequently  decanting  and  renewing  the  water,  a  thoroughly 
coherent  gluten  was  obtained.  This  was  washed  practically  starch  free  in 
a  current  of  water  and,  while  moist,  was  ground  by  passing  through  a 
special  form  of  '  '  drug-press,  '  '  which  was  a  ready  means  of  reducing  it  to 
comparatively  small  pieces.  The  ground  gluten  was  then  extracted  with 
alcohol  of  such  strength  that,  with  the  combined  water  of  the  gluten,  a 
solvent  of  60  to  70  per  cent  by  volume  resulted.  The  extracts  were  filtered 
perfectly  clear  through  thick  felts  of  filter  paper  pulp,  and  the  water-clear 
solution,  free  from  any  trace  of  opalescence  or  turbidity,  was  evaporated  to 
a  small  volume  on  a  water-bath.  The  thick  sirup  that  resulted  was  cooled 
and  then  poured,  with  constant  and  rapid  stirring,  into  a  large  volume  of 
distilled  ice-water  containing  a  very  little  sodium  chloride.  The  gliadin 
was  thus  precipitated  as  a  filament,  which,  on  stirring,  united  to  a  coherent 
plastic  mass.  This  gliadin  was  next  dissolved  by  stirring  with  strong  alcohol 
until  all  had  gone  into  solution,  the  water  combined  with  the  precipitated 
gliadin  being  sufficient  to  dilute  the  alcohol  to  the  proper  degree.  The 
resulting  solution  was  evaporated  to  a  thick  sirup,  absolute  alcohol  being 
added  from  time  to  time  in  order  to  hold  the  gliadin  in  the  solution,  since 
this,  during  the  evaporation,  became  constantly  more  aqueous.  The  thick 


^        EXPERIMENTAL.  71 

sirup  was  then  poured,  in  a  very  fine  stream,  into  a  large  volume  of  absolute 
alcohol  under  rapid  and  constant  stirring.  In  this  way  a  porous  mass  of 
protein  was  obtained  which  was  at  once  reduced  to  small  pieces  and  digested 
under  fresh  absolute  alcohol.  When  well  dehydrated,  the  gliadin  was 
digested  with  ether,  partially  dried  over  sulphuric  acid,  ground  to  a  coarse 
powder,  and  then  dried  thoroughly  over  sulphuric  acid.  When  thus  pre- 
pared, gliadin  forms  a  snow-white,  friable  mass  which  is  easily  reduced  to 
a  powder. 

1 1 oo  grams  of  gliadin,  equal  to  998.6  grams  dried  at  no0,  were  heated 
with  a  mixture  of  1000  cc.  of  concentrated  hydrochloric  acid  and  1000  cc. 
of  water  on  the  water-bath  for  several  hours,  until  the  gliadin  had  dissolved 
and  frothing  had  ceased.  The  solution  was  boiled  in  an  oil-bath  having  a 
temperature  of  1 15°  for  10  hours,  cooled  with  ice,  and  saturated  with  gaseous 
hydrochloric  acid.  After  remaining  on  ice  for  2  days,  the  glutaminic  acid 
hydrochloride  that  had  separated  was  filtered  out,  washed  with  ice-cold 
alcoholic  hydrochloric  acid  dissolved  in  water,  the  solution  treated  with 
bone-black  and  freed  from  ammonia  by  boiling  with  an  excess  of  barium 
hydroxide.  After  removing  the  barium  with  an  equivalent  amount  of  sul- 
phuric acid,  the  glutaminic  acid  was  separated  as  hydrochloride,  and,  when 
recrystallized  and  thoroughly  dried,  weighed  374.3  grams,  equivalent  to  300 
grams  of  free  glutaminic  acid.  This  was  converted  into  the  free  acid,  which 
melted  at  202°  to  203°. 

Carbon  and  hydrogen :  0.6218  gram  substance  gave  0.9276  gm.  CO,  and  0.3494  gm.  H2O. 
Nitrogen:  0.4574  gram  substance  gave  NH3  =  4.3  cc.  HC1  (i  cc.  HCl  =  o.oi  gram  N). 
Calculated  for  C5H9O4N,  C  40. 78,  H  6.18,  N  9.54  p.  ct.  ;  found,  C40.69,  H  6.24,  N  9.40  p.  ct. 

The  mother-liquor  from  the  recrystallized  glutaminic  acid  was  added  to 
the  filtrate  from  the  first  separation  of  the  glutaminic  acid,  and  the  entire 
solution  concentrated  to  a  sirup  under  strongly  reduced  pressure.  3  liters 
of  alcohol,  previously  saturated  with  hydrochloric  acid  at  a  low  temperature, 
were  added  to  the  sirup,  and  dry  hydrochloric  acid  gas  was  passed  into  the 
solution  until  it  was  saturated.  The  mixture  was  again  concentrated  as 
before,  under  reduced  pressure,  the  sirup  again  taken  up  in  3  liters  of  alco- 
holic hydrochloric  acid,  and  after  standing  several  hours  again  concentrated 
to  a  sirup,  taken  up  a  third  time  in  alcoholic  hydrochloric  acid,  and  after  some 
hours  concentrated  to  a  sirup  on  a  bath  of  40°  and  under  a  pressure  of  from 
5  to  10  mm.  The  neutralization,  extraction,  and  drying  of  the  esters  were 
conducted  according  to  the  method  described  by  Emil  Fischer.1 

The  undistilled  residue,  after  distillation  A,  weighed  180  grams. 

The  residue  which  remained  after  extracting  the  esters  with  ether  was 
made  strongly  acid  with  hydrochloric  acid,  freed  from  sodium  and  potassium 

1  Fischer,  Emil,  Zeitschrift  fur  physiologische  Chemie,  1901,  xxxin,  p.  151. 


72  THE    PROTEINS   OF   THE    WHEAT    KERNEL. 

salts  by  repeated  evaporations  with  alcoholic  hydrochloric  acid  and  thor- 
ough extraction  of  the  precipitated  chlorides  with  the  latter.  The  alcoholic 
solution  of  the  chlorides  of  the  amiuo-acids  was  evaporated  to  a  sirup,  and 
esterification  repeated  as  in  the  first  instance.  After  extracting  the  esters 
with  ether  and  drying  them,  the  entire  process  was  repeated,  and  the  ethe- 
real solution  of  the  esters  resulting  from  this  third  treatment  was  united 
with  that  from  the  second,  thus  following  the  method  applied  by  Abderhalden 
to  oxy hemoglobin.1 

Distillations  A  and  B. 


Fraction. 

Distillation  A. 

Distillation  B. 

Temper- 
ature of 
bath 
up  to  — 

Vapor. 

Press- 
ure. 

Weight. 

Temper- 
ature of 
bath 
up  to  — 

Press- 
ure. 

Weight. 

I  

o 

93 

IOO 

1  20 
1  60 

o 

75-76 

rum. 
I2.O 
I2.O 

0/8 
o.S 

Grams. 
28.18 
47-03 

64.  63 
40.00 

o 

83 

IOO 
120 
2OO 

mm. 
12.0 
12.  0 
0.8 

0.8 

Gtatns. 
20.13 
368. 
62.70 
33-00 

II  

Ill  

IV  

179.89 

152.64 

FRACTION  I. 

Distillation  A. — This  was  saponified  at  once  by  evaporation  with  con- 
centrated hydrochloric  acid  on  a  water-bath,  the  residue  taken  up  in 
alcohol,  the  solution  saturated  with  dry  hydrochloric  acid  gas,  and  a  crystal 
of  glycocoll  ester  hydrochloride  added.  After  prolonged  standing  on  ice, 
no  separation  occurred.  The  solution  was  then  evaporated  on  the  water- 
bath  with  concentrated  hydrochloric  acid,  the  latter  removed  with  lead  oxide 
.and  the  lead  with  hydrogen  sulphide.  The.  amino-acids  were  subjected  to 
fractional  crystallization. 

Distillation  B  was  treated  in  substantially  the  same  way,  but  although 
several  attempts  were  made  to  isolate  the  hydrochloride  of  glycocoll  ester, 
none  was  found.  By  systematic  fractionation  there  were  obtained  from 
fraction  I  of  the  two  distillations,  A  and  B,  6.68  grams  alanine  and  0.86 
gram  leucine. 

The  leucine,  when  recrystallized  from  dilute  alcohol,  decomposed  at 
about  298°. 

Carbon  and  hydrogen:  0.1778  gram  substance,  dried  at  no0,  gave  0.3577  gram  CO2  and- 

0.1608  gram  H2O. 
Calculated  for  C6Hi3O,N,  C  54.89,  H  10.01  p.  ct.;  found,  €34.87,  H  10.04  P-  ct. 


'Abderhalden,  Zeitschrift  fur  physiologische  Chemie,  1903,  xxxvn,  p.  484. 


EXPERIMENT  AL,. 


73 


The  alanine,  when  recrystallized  by  dissolving  in  a  little  hot  water  and 
gradually  adding  alcohol,  decomposed  at  about  290°. 

Carbon  and  hydrogen:  0.2404  gram  substance,  dried  at  110°,  gave  0.3571  gram  CO2  and 

0.1712  gram  H2O. 

Nitrogen:  o. 3980  gram  substance  gave  NH3  =  6. 2  cc.  HC1  (i  cc.  HCl  =  o.oi  gram  N). 
Calculated  for  C3H7O2N,  C  40.40,  H  7.93,  N  15.75  p.  ct.;  found,   C  40.51,  H  7.91, 

N  15.58  p.  ct. 

f  Fraction  II.  Temperature  of  bath  up  to  100°. 
(Pressure,  12  mm.     Weight,  83.84  grams. 

Distillations  A  and  B. — Bach  was  saponified  by 
5  parts  of  water. 

The  solution  was  evaporated  to  dry  ness  under  fWuced^  i^HBure,  the  dried 
residue  boiled  up  with  absolute  alcohol,  and  13.37  gram^^ere  dissolved. 
This  solution  was  united  with  a  similar  one  obtained  from  fraction  III. 
The  substance,  insoluble  in  alcohol,  after  systematic  fractional  crystalliza- 
tion, gave — 

(I)  22.1  grams  of  leucine,  which  decomposed  at  about  298°  and  had  the 
following  composition  : 

Carbon  and  hydrogen:  0.3128  gram  substance,  dried  at  rio°,  gave  0.6310  gram  CO2  and 

0.2794  gram  H2O. 
Calculated  for  C6H13O2N,  C  54.89,  H  10.01  p.  ct.;  found,  C  55.01,  H  9.92  p.  ct. 

(II)  A  fraction  of  3.45  grams  which  by  fractional  crystallization  could 
not  be  further  separated  and  gave  results  on  analysis  which  agreed  best  for 
a  mixture  of  leucine  and  amino-valerianic  acid. 

Carbon  and  hydrogen :  0.2857  gram  substance,  dried  at  110°,  gave  0.5519  gram  CO2  and 

0.2577  gram  H2O. 
Calculated  for  equal  molecules  of  leucine  and  amino-valerianic  acid,  C  53.05,  H  9.74 

p.  ct.;  found,  C  52.68,  H  10.02  p.  ct. 

(III)  2.1  grams  substance  which  had^he  properties  and  composition  of 
amino-valerianic  acid. 


,  dried  at  110°,  gave  0.3957  gram  CO4 
tance,  dried  at  110°,  gave  0.8643  gram 


.;  found,  (I) 


.03,  H  9.82  p.  ct.; 


Carbon  and  hydrogen:  (I)  0.2115  gram  sub 

and  0.1870  gram  H2O ;  (II)  0.4597  gra: 

CO2  and  0.3956  gram  H2O. 
Calculated  for  C5HUO2N,  C  51.22,  H  9. 

(II)  C  51-27,  H  9.56  p.  ct. 

Specific  rotation. — Dissolved  in  20  per  cent  hydrochloric  acid?  («)  -^-.=^ 

+  25.79°.  E.  Fischer  and  Dorpinghaus1  found  +  25.9°  for  their  preparation 
from  horn,  and  Schulze  and  Winterstein*  found  +28.2°  and  +27.9°  for 
preparations  from  lupine  seedlings. 


1  Fischer  &  Dorpinghaus,  Zeitschrift  fur  physiologische  Chemie,  1902, 
*  Schulze  &  Winterstein,  ibid.,  1902,  xxxv,  p.  300. 


.  462. 


74  THE  PROTEINS  OF  THE  WHEAT  KERNEL. 

The  chlorine  was  removed  from  the  solution  used  for  determining  the 
specific  rotation  and  the  substance  racemized  by  heating  with  20  cc.  of 
water  and  7  grams  of  crystallized  barium  hydroxide  for  19  hours  in  an 
autoclave  at  175°.  The  barium  was  quantitatively  removed  with  sulphuric 
acid,  and  the  a-naphthyl-hydantoic  acid  prepared  according  to  the  directions 
of  Neuberg  &  Manasse.1  This  crystallized  in  long  needles  and  melted 
constantly  on  repeated  recrystallization  from  40  per  cent  alcohol  at  180° 
to  181°. 

Carbon  Wtofo'drogcn^.^ss  gram  substance,  dried  at  90°,  gave  0.7936  gram  CO2  and 
Calculated  for  C16H18O3N2,  C  67.07,  H  6.35  p.  ct;  found,  C  66.90,  H  6.56  p.  ct. 

By  racemizirrg  the  remaining  mixture  of  undetermined  amino-acids  we 
were  unable  to  isolate  any  more  amino-valerianic  acid. 

(IV)  8.6  grams  alanine.  This  was  racemized  by  heating  with  an  excess 
of  barium  hydroxide  and  coupled  with  «-naphthyliso-cyanate  according  to  the 
directions  of  Neuberg  &  Manasse.  The  hydantoic  acid,  which  crystal- 
lized in  prisms,  melted  at  197°. 

Carbon  and  hydrogen:  0.3146  gram  substance,  dried  at  110°,  gave  0.7480  gram  CO2  and 

o.  1560  gram  H2O. 
Calculated  for  CUHUO3N2,  C  65.06,  H  5.48  p.  ct.;  found,  C  64.84,  H  5.51  p.  ct. 

f  Fraction  III.    Temperature  of  bath,  up  to  120°.  ) 
(Pressure,  0.8  mm.     Weight,  127.38  grams.       j 

This  fraction  was  boiled  for  5^  hours  with  8  parts  of  water.  The  solution, 
evaporated  to  dryness  under  reduced  pressure,  gave  98  grams  of  amino-acids 
or  80  per  cent  of  the  esters.  Of  this  59.94  grams  were  soluble  in  alcohol. 
From  the  part  insoluble  in  alcohol,  by  systematic  fractional  crystallization, 
there  were  isolated  33.06  grams  of  leucine  and  4.57  grams  of  alanine.  The 
leucine  decomposed  at  about  298 

Carbon  and  hydrogen  :  0.2416  gra^^fcbstance,  dried  at  110°,  gave  0.4872  gram  CO2 

and  0.2166  gram  II,  O. 

Nitrogen  :  0.2690  gram  substance  gjHs  =  2.85  cc.  HC1  (i  cc.  HCl  =  o.oi  gram  N). 
Calculated  ft>r  C6HlsO.jN,  C  54.89,  Bw",  N  10.70  p.  ct  ;  found,  C  54-99,  H  9.96, 

N  10.59  P- 


The  alcohol-  soluble  substance        n  fraction  II  was  united  with  that  from 

fraction  III.     The  solution  was  evaporated  to  dryness  under  reduced  press- 

ure, and  the  residue  taken  up  in  water  and  boiled  gently  for  about  an  hour 

1  Neuberg  &  Manasse,  Berichte  der  deutschen  chemischen  Gesellschaft,  1905,  xxxvm, 
P-  2359- 


EXPERIMENTAL.  75 

with  an  excess  of  copper  hydroxide.  The  filtered  solution  was  evaporated 
to  dryness  under  reduced  pressure  and  the  residue  boiled  with  absolute 
alcohol.  The  undissolved  part  was  dissolved  in  water,  freed  from  copper  by 
hydrogen  sulphide,  and  the  solution  again  evaporated  to  dryness  under 
reduced  pressure.  The  residue  was  boiled  with  absolute  alcohol,  in  which 
all  of  it  dissolved.  The  alcohol  was  evaporated  off  under  reduced  pressure,. 
the  residue  dissolved  in  500  cc.  of  water  and  again  converted  into  the  copper 
salt.  By  concentration  19.91  grams  of  crystalline  racemic  proline  copper 
salt  were  obtained,  which  is  equal  to  13.98  grams  of  a-proline.  This  was 
recrystallized  from  water  and  dried  in  the  air. 

Water  :  0.7789  gram  substance  lost  0.0856  gram  H2O  at  110°. 

Calculated  for  C10H16O4N2  Cu  •  2H2O,  H2O  11.00  p.  ct.  ;  found,  H2O  10.98  p.  ct. 

Carbon  and  hydrogen  :  0.6851  gram  substance,  dried  at  110°,  gave  1.0310  gram  CO2  and 

0.345°  gram  H2O. 

Copper:  0.2926  gram  substance  gave  0.0799  gram  CuO. 
Calculated  for  Ci0H16O4N2  Cu,  C  41.11,  H  5.54,  Cu  21.79  P-  ct  '>  found,  C  41.04,  H  5.59, 

Cu  21.  81  p.  ct. 

The  r-proline  copper  salt  was  freed  from  copper  with  hydrogen  sulphide, 
its  solution  evaporated  to  dryness,  and  the  residue  recrystallized  from  alcohol. 
After  drying  in  vacuo  over  sulphuric  acid  the  r-proline  melted  at  203°  to 

205V 

Carbon  and  hydrogen:  0.3373  gm.  substance  gave  0.6424  gm.  CO2  and  0.2453  gm«  H2O. 
Calculated  for  C5H9O2  N,  C  52.12,  H.  7.90  p.  ct.  ;  found,  C  51.94,  H  8.08  p.  ct. 

The  solution  of  the  alcohol-soluble  copper  salt  was  evaporated  to  dry- 
ness  and  left  a  residue  of  /-proline  copper  salt  which,  dried  at  120°,  weighed 
71.62  grams,  which  is  equal  to  56.51  grams  of  free  /-a-proline. 

Copper:  0.2850  gram  substance,  dried  at  110°,  gave  0.0760  gram  CuO. 
Calculated  for  C10H16O4N2  Cu,  Cu  21.79  P-  ct.  J  found,  Cu  21.30  p.  ct. 

One-half  of  this  proline  copper  salt  was  freed  from  copper,  and  the  pro- 
line  racemized  by  heating  with  150  cc.  of  water  containing  80  grams  of 
crystallized  barium  hydroxide  for  5  hours  at  150°.  The  barium  was  re- 
moved quantitatively  with  sulphuric  acid,  the  solution  concentrated,  and  the 
proline  again  converted  into  the  copper  salt.  There  was  thus  obtained  20.  r 
grams  of  very  nearly  pure  racemic  a-proline  copper. 

Water  :  0.3356  gram  substance,  air-dry,  lost  0.0369  gram  H2O  at  110°. 
Copper  :  0.2640  gram  substance  gave  0.0640  gram  CuO. 

Calculatedfor  C10H16O4N,  Cu  •  2H2O,  H2O  11.00,  Cu  19.40  p.  ct.  ;  found,  H2O  11.00,  Cu 
P-  ct. 


1  Willstaeter,  R.,  Berichte  der  deutschen  chemischen  Gesellschaft,  1900,  xxxm,  p.  1160;. 
Fischer,  Emil,  ibid.,  1901,  xxxiv,  p.  458. 


76 


THE;  PROTEINS  OF  THE  WHEAT  KERNEL. 


The  total  crystalline  racemic  copper  salt  was  equal  to  41.96  grains  of 
r-a-proline. 

From  the  other  half  of  /-proline  copper  salt  the  free  proline  was  regen- 
erated and  recrystallized  from  alcohol.  A  small  part  only  was  obtained  in 
a  crystalline  condition,  which  melted  at  205°  to  206°. 

Carbon  and  hydrogen :  0.2729  gram  substance,  dried  over  H2SO4)  gave  0.5198  gram 

CO2  and  0.1977  gram  H2O. 

Nitrogen  :  0.2453  gram  substance  gave  NH3  =  2.95  cc.  HC1  (i  cc.  HC1  =  o.oi  gram  N). 
Calculated  for  C5H9O2N,  C  52.12,  H  7.90,   N  12.20  p.  ct.  ;  found,  C  51.95,  H  804, 

N.  12.03  P-  ct. 

From  fraction  III  there  were  isolated  4.57  grams  alanine,  33.06  grams 
leucine,  and  70.49  grams  a-proline,  including  in  this  last  that  from  frac- 
tion II,  which  was  not  weighed  separately. 


Fraction  IV. 

Temper- 
ature 
of  bath 
to  — 

Press- 
ure. 

Weight. 

A  

0 

160 

mm. 

0.8 

Grams. 
-jq  q6 

B  

200 

0.8 

T.1  OO 

72.Q6 

1 

This  was  treated  with  water  and  shaken  out  with  ether  according  to  the 
procedure  described  by  Emil  Fischer.1 

The  ether  was  carefully  removed  by  evaporation  and  the  residual  phenyl- 
alanine  ester  saponified  by  dissolving  in  concentrated  hydrochloric  acid 
and  evaporating  on  a  water-bath.  The  phenylalanine  hydrochloride  weighed 
29. 14  grams,  equivalent  to  23.87  grams  of  free  phenylalanine.  The  phenyl- 
alanine hydrochloride  was  recrystallized  from  strong  hydrochloric  acid. 
It  was  decomposed  by  evaporating  with  an  excess  of  ammonia  and  the 
phenylalanine  recrystallized  from  water.  It  melted,  on  slow  heating,  at 
263°  to  265°. 2 

Carbon  and  hydrogen  :  0.3051  gram  substance,  dried  at  110°,  gave  0.7322  gram  CO2  and 

0.1792  gram  HjO. 

Nitrogen :  0.3020  gram  substance  gave  NH3=  2.53  cc.  HC1  (i  cc.  HC1  =  o.oi  gram  N). 
Calculated  for  C9HUO,N,  C  65.40,  H  6.73,  N  8.50  p.  ct.  ;  found,  C  65.44,  H  6.53,  N  8.38 

p.  ct. 

Fischer,  E.,  Zeitschrift  fiir  physiologische  Chemie,  1902,  xxxvi,  p.  274. 
*  Fischer,  Emil,  &  Abderhalden,  Zeitschrift  fiir  physiologische  Chemie,  1902,  xxxvi, 
p.  268  ;  Erlenmeyer  &  kipp,  Annalen  der  Chemie,  1883,  ccxix,  p.  197. 


EXPERIMENTAL.  77 

The  aqueous  layer  was  heated  with  an  excess  of  barium  hydroxide  on  a 
water-bath  for  5  hours.  After  standing  some  time,  the  barium  salt  that  had 
separated  was  filtered  out  and  decomposed  by  an  equivalent  amount  of  sul- 
phuric acid.  The  solution  on  concentration  yielded  5.76  grams  of  aspartic 
acid. 

Carbon  and  hydrogen :  0.3866  gram  substance,  dried  at  110°,  gave  0.5 109  gram  CO2  and 

0.1852  gram  H2O. 

Nitrogen  :  0.3637  gram  substance  gave  NH3  =  3.78  cc.  HC1  (i  cc.  HC1  =  o.oi  gram  N). 
Calculated  for  C4H7O4N,  C  36.09,  H  5.26,  N  10.53  P-  ct.  ;  found,  C  36.04,  H  5.32,  N 

10.39  P-  ct- 

The  filtrate  from  the  barium  aspartate  was  freed  from  barium,  concen- 
trated to  small  volume,  and  saturated  with  hydrochloric  acid.  On  prolonged 
standing  a  trace  of  phenylalanine  hydrochloride  separated,  but  no  glutaminic 
acid  hydrochloride  was  obtained.  After  removing  the  hydrochloric  acid 
with  silver  sulphate  and  the  sulphuric  acid  with  barium  hydroxide,  the 
solution  was  boiled  with  an  excess  of  copper  hydroxide,  but  no  copper  salt 
could  be  separated  from  it,  even  after  concentrating  to  a  very  small  volume. 
The  copper  was  then  removed  and  the  solution  treated  with  bone-black  and, 
when  concentrated  in  a  vacuum  over  sulphuric  acid,  gave  crystals  which,  on 
fractional  crystallization  from  water,  gave  0.42  gram  of  serine,  which,  in 
an  open  capillary,  browned  at  about  218°  and  decomposed  to  a  brownish 
mass  at  about  240.  ° 

Carbon  and  hydrogen:  0.2577  gram  substance,  dried  at  110°,  gave  0.3224  gram  CO, 

and  0.1602  gram  H2O. 
Calculated  for  C3H,O3N,  C  34.24,  H  6.73  p.  ct.  ;  found,  C  34.12,  H  6.91  p.  ct. 

In  the  filtrate  from  the  serine  there  was  obtained  about  5.32  grams  of  crys- 
talline substance,  from  which  nothing  definite  could  be  isolated. 

DISTILLATION  RESIDUE. 

The  residues  from  distillations  A  and  B  were  dissolved  in  boiling  alcohol 
and  the  solutions  united.  On  cooling,  5.76  grams  of  long  hair- like  crystals 
separated. 

The  filtrate  from  this  substance  was  freed  from  alcohol  and  saponified  by 
heating  with  200  grams  of  crystallized  barium  hydroxide,  the  barium  re- 
moved, the  solution  concentrated  under  reduced  pressure  to  small  volume, 
saturated  with  hydrochloric  acid,  and,  after  standing  on  ice  for  some  time, 
yielded  74.21  grams  glutaminic  acid  hydrochloride,  equal  to  59.46  grams.1 

The  glutaminic  acid  hydrochloride  melted  at  about  198°  with  effervescence. 

Carbon  and  hydrogen :  0.4028  gram  substance,  dried  over  H2SO5,  gave  0.4802  gram 

CO2  and  0.2056  gram  H2O. 
Calculated  for  C5H10O4  NCI,  C  32.67,  H  5.50  p.  ct.  ;  found,  C  32.51,  H  5.67  p.  ct. 

'Abderhalden  &  Wells,  Zeitschrift  fur  physiologische  Chemie,  1905,  XLVI,  p.  31. 


THE    PROTEINS   OF   THE)    WHEAT    KERNEL. 


20L 


Specific  rotation. — Dissolved  in  20  per  cent  hydrochloric  acid,  (a)  —  = 

-I-  31-47°. 

Fischer  &  Dorpinghaus  found  +31.91°  for  a  preparation  from  horn, 
-{-30.45°  from  gelatin,  and  +28.20°  for  one  from  casein.1 

The  residue  which  remained  after  removing  the  esters  with  ether  from 
the  original  solution  of  the  products  of  hydrolysis  was  treated  in  the 
way  described  by  Emil  Fischer2  for  the  isolation  of  oxy-proline.  The 
only  substance,  however,  that  could  be  isolated  was  serine,  of  which 
0.87  gram  was  obtained,  which  browned  at  about  219°  and  decomposed  at 
about  240°. 

Carbon  atid  hydrogen:  0.2987  gram  substance,  dried  at  110°,  gave  0.3743  gram  CO2 

and  0.1844  gram  H2O. 
Calculated  for  C3H7O3N,  C  34.24,  H  6.73  p.  ct.  ;  found,  C  34.18,  H  6.85  p.  ct. 

In  the  filtrate  from  this  serine  /3-naphthalene-sulphone  chloride  failed  to 
give  any  definite  product. 

There  were  thus  isolated  in  the  two  distillations  from  fraction  I,  6.68 
grams  ;  from  fraction  II,  8. 59  grams,  and  from  fraction  III,  4.57  grams  of 
alanine,  or  19.84  grams  in  all;  from  fraction  II,  2.1  grams  of  amino-vale- 
rianic  acid  ;  from  fraction  I,  0.86  gram  ;  from  fraction  II,  22. 10  grams  ;  from 
fraction  III,  33.06  grams  of  leucine — in  all  56.02  grams  ;  from  fractions  II 
and  III,  70.49  grams  of  proline  ;  from  fraction  IV,  23.47  grams  of  phenyl- 
alanine,  5.76  grams  of  aspartic  acid,  and  0.42  grams  of  serine,  and  from  the 
residue  which  remained  after  extracting  the  esters  0.87  gram  of  serine,  or 
1.29  grams  in  all. 

From  the  main  solution  of  the  total  products  of  hydrolysis  there  were 
obtained  300  grams  of  glutaminic  acid,  and  from  the  residues  after  dis- 
tilling the  esters  59.5  grams,  or  in  all  359.5  grams. 

As  the  amount  of  proline  found  in  this  hydrolysis  was  so  great,  the  result 
was  confirmed  by  a  second  hydrolysis,  and  another  effort  made  to  obtain 
glycocoll,  which,  if  present  in  very  small  amount,  might  have  escaped 
detection. 

For  this  purpose  500  grams  of  gliadin,  air-dry,  equal  to  439.6  grams  dried 
at  110°,  were  hydrolyzed  in  the  same  way  as  in  the  preceding  hydrolysis. 
After  esterifying  and  shaking  out  the  esters  three  times,  as  before,  the  ether 
was  removed  by  distillation  at  760  mm.  and  the  esters  distilled. 

JCf.  Fischer,    Emil,  &  Dorpinghaus,  Zeitschrift  fur  physiologische   Chemie,    1902, 
xxxyi,  p.  475. 
2  Fischer,  Emil,  Berichte  der  deutschen  chemischen  Gesellschaft,  1902,  xxxv,  p.  2660 


EXPERIMENTAL. 


79 


Fraction. 

Tempera- 
ture of 
bath 
up  to  — 

Pressure. 

Weight. 

I  

o 
qc 

mm. 
I7.oo 

Grams. 
2O.2O 

II  

80 

S.oo 

46.08 

™  {$ 

IV  

IIO 

no 
180 

2.00 

0.88 
0.88 

44.14 

15.70 
1:4.70 

V  

200 

0.78 

2Q.6q 

2IO.5I 

Fraction  I  was  immediately  evaporated  on  the  water-bath  with  hydro- 
chloric acid,  the  residue  dissolved  in  alcohol,  and  the  solution  saturated  with 
dry  hydrochloric  acid  gas.  The  solution  was  concentrated  to  a  small  volume 
at  a  low  temperature  under  a  pressure  of  10  mm.,  the  residue  taken  up  in 
alcohol,  its  solution  cooled  to  o°  and  saturated  with  hydrochloric  acid  gas. 
On  prolonged  standing  0.22  gram  of  glycocoll  ester  hydrochloric  separated, 
which  melted  at  144°  to  145°.  When  mixed  with  pure  glycocoll  ester 
hydrochloride,  the  melting-point  was  unchanged. 

Chlorine :  0.1058  gram  substance  gave  o.  1063  gram  AgCl  =  Cl  24.86. 
Calculated  for  C4H10O2NC1,  Cl  25.40  p.  ct 

Neither  fraction  II  nor  the  ether  distilled  from  the  esters  gave  evidence  of 
glycocoll. 

This  preparation  of  gliadin  did,  in  fact,  contain  a  very  small  amount  of 
glycocoll,  which  is  possibly  due  to  a  slight  contamination  with  glutenin,  in 
which  there  has  since  been  found  a  notable  quantity  of  this  amino-acid. 

Fraction  II  was  saponified,  and  the  solution  evaporated  under  highly 
reduced  pressure  from  a  bath  at  40°,  and  the  residue  extracted  with  alcohol, 
in  which  about  18  grams  dissolved. 

Fraction  III,  by  similar  treatment,  yielded  24  grams  of  alcohol-soluble 
substance.  The  alcoholic  solutions  were  united  and  evaporated  to  dryness 
from  a  bath  at  40°.  The  crystalline  residue,  when  dried  to  constant  weight 
in  vacuo,  weighed  39.59  grams.  From  this,  by  extraction  with  alcohol,  8.7 
grams  of  substance  insoluble  therein  were  separated.1  The  total  proline 
thus  found  was  30.89  grains,  equal  to  7.03  per  cent. 

1  Emil  Fischer  employs  this  method  for  estimating  the  proportion  of  proline  in  pro- 
teins, but  stated  that  the  result  obtained  is  too  high  (Berichte  der  deutschen  chemischen 
Gesellschaft,  1906,  xxxix,  p.  530). 


8O  THE   PROTEINS    OE   THE    WHEAT    KERNEL. 

CYSTINE. 

300  grams  of  gliadin  were  digested  for  2  to  3  hours  at  85°  with  900  cc.  of 
hydrochloric  acid,  sp.  gr.  1.19,  and  the  solution  boiled  for  3  hours.  This 
was  then  concentrated  to  a  sirup  under  diminished  pressure,  diluted  to  900 
cc.  with  cold  water,  and  neutralized  with  50  per  cent  sodium-hydroxide  solu- 
tion. After  boiling  with  a  large  amount  of  bone-black  and  concentrating  to 
800  cc.,  much  substance  separated,  which  was  recry stall ized  from  about  300 
cc.  of  water.  The  recrystallized  product  was  dissolved  in  5  per  cent  sul- 
phuric acid  and  precipitated  by  mercuric  sulphate  solution.1 

The  mercury  precipitate  was  decomposed  by  hydrogen  sulphide,  the  solu- 
tion freed  from  hydrogen  sulphide,  neutralized  with  sodium  hydroxide,  and 
acidified  with  acetic  acid.  On  standing,  cystine  separated  from  the  solution 
in  hexagonal  plates,  and  by  adding  alcohol  to  the  filtrate  more  was  obtained. 
When  no  more  cystine  could  be  thus  obtained,  the  precipitation  with  mercu- 
ric sulphate  was  repeated.  By  several  repetitions  of  this  process  1. 18  grams 
of  cystine  were  finally  isolated,  which,  when  recrystallized  by  dissolving  in 
dilute  ammonia  and  acidifying  with  acetic  acid,  gave  the  following  analysis  : 

Carbon  and  hydrogen :  0.3063  gram  substance,  dried  at  110°,  gave  0.3379  gram  CO2  and 
0.1444  gram  H2O. 

Calculated  for  CgH^O^S-j,  C  29.96,  H  5.04  p.  ct.  ;  found,  C  30.08,  H  5.23  p.  ct. 

TYROSINE. 

219  grams  of  gliadin,  equal  to  200  grams  dried  at  no0,  were  treated  with 
600  cc.  of  concentrated  hydrochloric  acid,  digested  for  some  time  on  a  water- 
bath,  and  the  solution  boiled  for  12  hours  on  an  oil-bath.  The  solution  was 
freed  from  most  of  the  glutaminic  acid  by  saturating  with  hydrochloric  acid, 
and  the  filtrate  from  the  glutaminic  acid  was  diluted,  boiled  with  bone-black, 
and  then  concentrated  strongly  to  remove  as  much  hydrochloric  acid  as 
possible.  The  rest  of  the  acid  was  neutralized  with  50  per  cent  sodium- 
hydroxide  solution.  On  standing,  a  considerable  precipitate  separated, 
which  was  filtered  out  and  dissolved  in  ammonia.  The  resulting  solution 
was  boiled  until  most  of  the  ammonia  had  been  removed  and  the  tyrosine 
that  separated  was  filtered  out.  When  dried,  this  weighed  2.4  grams,  equal 
to  1.2  per  cent  of  the  gliadin.  Recrystallized  from  boiling  water,  this  gave 
the  following  results  on  analysis  : 

Carbon  and  hydrogen:  0.3661  gram  substance,  dried  at  no°,gave  0.7981  gram  CO2  and 

0.2160  gram  H,O. 
Calculated  for  C9HUO3N,  C  59.62,  H  6.13  p.  ct.  ;  found,  C  59.45,  H  6.56  p.  ct. 

Tyrosine  separated  from  our  hydrolysis  solutions  of  gliadin  with  very 
great  difficulty.  Two  other  attempts  to  determine  its  proportion,  which 
were  made  by  hydrolyzing  with  sulphuric  acid,  gave  lower  results,  and  the 

1  Cf.  Hopkins  &  Cole,  Journal  of  Physiology,  1901,  xxvil,  p.  418. 


EXPERIMENTAL.  81 

solutions  from  which  the  tyrosine  separated  still  continued  to  give  a  strong 
Millon's  reaction.  Kutscher1  found  2.09  per  cent  of  tyrosine  in  gliadin, 
and  Abderhalden  &  Samuely'2  found  2.37  per  cent. 

HISTIDINE. 

Fifty  grams  of  gliadin,  equal  to  43.97  grams  dried  at  1 10° ,  were  hydrolyzed 
according  to  the  directions  of  Kossel  &  Kutscher3  and  the  determination 
of  the  bases  carried  out  according  to  the  method  of  Kossel  &  Patten.* 

The  solution  of  the  histidine  was  made  up  to  500  cc.  and  nitrogen  deter- 
mined in  100  cc.  of  it. 

TOO  cc.  of  solution  gave  ammonia  =  1.37  cc.  HC1  (i  cc.  HC1  =  o.oi  gram  N)  =  0.0137 
gram  N  =  0.0685  gram  N  in  500  cc.  =  0.2524  gram  histidine  =  0.58  p.  ct.  of  the 
gliadin. 

The  identity  of  this  histidine  was  not  established,  as  the  quantity  was  too 
small  to  permit  the  preparation  of  a  satisfactory  product. 

ARGININE. 

The  arginine  solution  was  made  up  to  500  cc.  and  nitrogen  determined  in 
50  cc.  of  it. 

50  cc.  of  solution  gave  ammonia  =4. 15  cc.  HC1  (i  cc.  HCl  =  o.oi  gram  N)  =  0.0415 
gram  N  or  0.415  gram  in  500  cc.  =  1.39  gram  arginiue  or  3.16  p.  ct.  of  the  gliadin. 

The  remaining  solution  was  treated  as  Kossel  &  Kutscher  direct  and  the 
argicine  obtained  as  carbonate.  A  portion  of  this  carbonate  was  converted 
into  the  picrolonate  according  to  the  directions  of  Steudel.5  This  melted  at 
226°  to  227°,  while  Steudel  gives  225°. 

Nitrogen:  0.0832  gram  substance,  dried  at  110°,  gave  18.8  cc.  moist  N2  at  765  mm. 

and  25°. 
Calculated  for  C6HUO2N4  •  C10H8O5N4,  N  25.62  p.  ct.;  found,  N  25.40  p.  ct. 

LYSINE. 

The  filtrate  from  the  silver  precipitate  of  arginine  and  histidine  was  freed 
from  silver,  precipitated  with  phosphotungstic  acid,  and  lysine  tested  for 
with  picric  acid  in  the  usual  way,  but  none  was  found. 

Kossel  &  Kutscher6  found,  in  the  three  fractions  of  the  alcohol-soluble 
protein  of  the  wheat  kernel  which  they  examined,  1.2,  0.43,  and  1.53  per 
cent  of  histidine.  The  writer' s  determination  falls  between  these .  Kutscher, 7 
in  discussing  the  individuality  of  these  three  fractions,  considers  the  differ- 

1  Kutscher,  Zeitschrift  fur  physiologische  Chemie,  1903,  xxxvni,  p.  in. 

2  Abderhalden  &  Samuely,  ibid.,  1905,  XLIV,  p.  276. 
3 Kossel  &  Kutscher,  ibid.,  1900,  xxxi,  p.  165. 

4 Kossel  &  Patten,  ibid.,  1903,  xxxviu,  p.  39. 
5 Steudel,  ibid.,  1902,  xxxvn,  p.  219. 
6  Kossel  &  Kutscher,  loc.  cit. 
T  Kutscher,  loc.  cit. 
6 


82  THE   PROTEINS    OF   THE    WHEAT    KERNEL. 

ences  in  the  amount  of  histidine  found  in  them  to  be  within  the  limits  of 
accuracy  of  these  determinations. 

Kossel  &  Kutscher  also  found  3.05  per  cent  of  arginine  in  the  fraction 
which  they  called  gluten-fibrin,  2.75  per  cent  in  their  gliadin,  and  3.13  per 
cent  in  their  mucedin.  In  determining  the  amount  of  protein  hydrolyzed 
they  calculated  the  weight  from  the  nitrogen  in  solution.  If,  as  seems 
probable,  only  one  alcohol-soluble  protein  exists  in  this  seed,  namely, 
gliadin,  with  17.5  per  cent  of  nitrogen,  and  that  the  nitrogen  of  their  solu- 
tions belonged  to  this  protein,  the  proportion  of  arginine  found  by  them 
would  be,  respectively,  3.13,  2.79.  and  3.25  percent,  with  which  3.16  per 
cent  agrees  very  closely.  The  results  of  this  hydrolysis  are  given  in  table  1 7. 

TABLE  17. — Gliadin. 


P.ct. 

Glycpcoll o.oo 

Alanine 2.00 

Amino-valerianic  acid 0.21 

Leucine 5.61 

a-proline 7.06 

Phenylalanine 2.35 

Aspartic  acid 0.58 

Glutaminic  acid 37-33 

Serine o.  13 

Tyrosine 1.20 


P.ct, 

Cystine 0.45 

Oxy-proline 

Lysine o.oo 

Histidine 0.58 

Arginine 3.16 

Ammonia 5.11 

Tryptophane present 

65.78 


PROTEIN   INSOLUBLE   IN   WATiSR,    SALINE  SOLUTIONS,    AND   ALCOHOL — 

GLUTENIN. 

As  already  stated,  extraction  with  the  above-named  solvents,  applied  suc- 
cessively, removed  but  a  part  of  the  total  protein  contained  in  the  wheat 
kernel,  that  remaining  being  soluble  only  in  dilute  acids  and  alkalis.  The 
following  extractions  were  next  made  to  determine  the  nature  of  this  body. 

PROTEIN  EXTRACTED  BY  DILUTE  ALKALINE  SOLUTIONS  AFTER  EXTRACTING  THE 
FLOUR  WITH  10  PER  CENT  SODIUM-CHLORIDE  BRINE  AND  THEN  WITH  DILUTE 
ALCOHOL. 

After  completely  extracting  from  4000  grams  "straight  flour"  all  the 
protein  soluble  in  10  per  cent  sodium  chloride  solution,  the  residue  was 
freed  wholly  from  protein  soluble  in  cold  alcohol  of  0.90  sp.  gr.  The  re- 
sulting residue  was  then  extracted  twice  with  a  large  quantity  of  o.  i  per 
cent  potassium  hydroxide  solution.  After  standing  3  days  at  a  tempera- 
ture of  5°  with  frequent  stirring,  this  extract  was  filtered  off  and  allowed 
to  stand  in  a  cold  room  until  the  greater  part  of  the  fine  starch  and  other 
impurities  which  had  escaped  filtration  had  settled.  The  solution,  which 
was  still  turbid,  was  decanted  from  the  sediment  and  neutralized  as  exactly 
as  possible  with  0.2  per  cent  hydrochloric  acid,  thereby  producing  a  precipi- 


EXPERIMENTAL. 


tate  which  settled  rapidly,  leaving  the  filtrate  milky  in  appearance.  This 
precipitate,  after  decanting  the  solution,  was  dissolved  in  0.2  per  cent  potas- 
sium-hydroxide water  and  set  aside  to  deposit  the  suspended  impurities. 
After  decantation  from  the  sediment  which  resulted,  the  solution  was  filtered, 
but  almost  nothing  was  thus  removed.  It  was  then  neutralized  with  0.2 
per  cent  hydrochloric  acid  and  the  precipitate  produced  washed  by  decan- 
tation, first  with  water  and  then  with  dilute  alcohol,  absolute  alcohol,  and 
ether.  No  attempt  was  made  in  this  case  to  obtain  the  whole  of  the  protein 
soluble  in  alkali,  as  the  difficulties  presented  by  slow  and  imperfect  filtration 
rendered  this  impossible.  The  substance  thus  obtained  formed  a  brownish, 
horny  mass,  which  weighed  13  grams.  This  preparation,  89,  when  dried 
at  110°,  on  analysis  gave  the  results  shown  in  the  table  at  the  bottom  of  this 
page. 

Subsequent  preparations  of  this  body  led  to  the  idea  that  it  was  far  from 
pure,  this  supposition  being  supported  by  the  fact  that  the  final  solution 
from  which  it  had  been  precipitated  was  turbid.  The  remainder  of  the 
preparation  was  then  dissolved  as  completely  as  possible  in  0.2  per  cent 
potassium-hydroxide  solution,  and  by  repeated  filtration  through  very  dense 
filter-paper  obtained  perfectly  clear.  A  considerable  insoluble  residue 
remained,  which  appeared  to  consist  largely  of  the  coagulated  form  of  this 
protein.  This  residue  was  washed  by  decantation  with  water,  alcohol,  and 
ether.  It  was  found  to  contain  but  13.68  per  cent  of  nitrogen,  showing 
that  the  preparation,  89,  contained  much  non-nitrogenous  matter.  The  filtra- 
tion of  the  dissolved  protein  proceeded  very  slowly,  so  that  it  was  conducted 
at  a  temperature  near  o° .  A  portion  of  the  filtrate  first  obtained  was  re- 
moved, precipitated  by  0.2  per  cent  hydrochloric  acid,  washed  with  water, 
alcohol,  and  ether,  and  the  preparation,  90,  analyzed  with  the  following 
results.  A  second  portion  similarly  yielded  preparation  91. 

Preparations  89,  go,  and  pi. 


Preparation  89. 

Preparation  90. 

Preparation  91. 

I. 

Ash-free. 

I. 

II. 

Average. 

Ash-free. 

I. 

Ash-free. 

Carbon  
Hydrogen  . 
Nitrogen... 
Sulphur  
Oxygen  — 
Ash  

P.ct. 
52.47 
6-75 
I5.5I 
0.86 

0.88' 

P.  ct. 

52.91 
6.81 

15-65 
0.86 

23-77 

P.  ct. 
51.10 
6.46 
17.01 
0.91 

2.28 

P.ct. 

P.ct. 
51.10 
6.46 
17.01 
0.92 

P.ct. 
52.29 
6.61 

17.41 
0.94 
22.75 

P.  ct. 

P.ct. 

17.17 

17-33 

0.92 

0.97 



IOO.OO 







IOO.OO 





84 


THE;  PROTEINS  OF  THE;  WHEAT  KERNEL. 


These  results  show  that  it  is  absolutely  necessary  to  filter  the  alkaline 
solution  of  this  body  perfectly  clear  before  the  final  precipitation,  since  other- 
wise a  considerable  amount  of  non-nitrogenous  matter  will  be  precipitated 
with  it.  Another  extraction  was  made  by  treating  200  grams  of  "  patent 
flour  "  from  spring  wheat  with  10  per  cent  sodium-chloride  solution  added 
in  small  quantities  so  as  to  make  a  dough.  This  dough  was  then  washed 
with  10  per  cent  sodium-chloride  brine  until  nearly  all  the  starch  was  re- 
moved and  a  gluten  obtained  similar  in  all  respects  to  that  resulting  from 
treating  the  flour  with  water.  This  gluten  was  then  chopped  fine,  thoroughly 
extracted  with  alcohol  of  sp.  gr.  0.90,  and  then  dissolved  at  20°  in  o.i  per 
cent  potassium-hydroxide  water.  The  -resulting  solution  was  filtered,  but, 
as  only  a  part  of  the  impurities  was  thus  removed,  the  filtrate  was  placed  in 
an  ice-box  in  shallow  dishes  and  allowed  to  deposit  a  considerable  part  of 
the  suspended  matter.  The  decanted  solution,  which  was  only  very  slightly 
turbid,  was  then  precipitated  by  neutralizing  with  0.2  per  cent  hydro- 
chloric acid,  and  the  precipitate  washed  by  decantation  with  water,  extracted 
thoroughly  with  dilute  alcohol,  digested  with  absolute  alcohol  and  then  with 
ether.  This  preparation,  92,  was  a  snow-white,  light,  porous  mass,  easily 
reduced  to  a  powder.  It  had  the  following  composition  : 

Preparation  92. 


I. 

II. 

Average. 

Ash-free. 

Carbon  

P.ct. 

P.ct 

52.18 

P.ct. 

P.  ct. 
52.50 

6.QO 

6.04 

Nitrogen  

17.11 

16.90 

17.01 

17.22 

Sulphur  

I.OO 

I.OO 

22.14 

Ash 

O.6* 

IOO.OO 

PROTEIN  EXTRACTED  BY  DILUTE  ALKALINE  SOLUTIONS  FROM  GLUTEN  AFTER 
EXTRACTING  THE  LATTER  WITH  DILUTE  ALCOHOL. 

2000  grams  of  ' '  straight  flour ' '  from  spring  wheat  were  made  into  a  dough 
with  distilled  water,  and  this  was  washed  with  river- water  until  the  gluten 
was  freed  as  completely  as  possible  from  starch.  This  gluten  was  then  ex- 
tracted with  75  per  cent  alcohol  as  long  as  anything  was  removed.  The 
insoluble  residue  was  dissolved  in  0.15  per  cent  potassium-hydroxide  water 
and  the  resulting  solution  allowed  to  stand  in  a  cold  room  for  48  hours. 
The  solution  was  thus  freed  from  but  a  part  of  the  suspended  matter.  After 
decanting  from  the  sediment,  the  solution  was  neutralized  with  dilute  hydro- 
chloric acid,  the  precipitate  produced,  washed  several  times  by  decantation 


EXPERIMENTAL. 


with  water,  thoroughly  extracted  with  alcohol  of  0.90  sp.  gr.,  then  with 
stronger  alcohol,  and  finally  with  absolute  alcohol  and  with  ether. 

The  precipitate  was  again  dissolved  in  o.  i  per  cent  potassium-hydroxide 
solution  and  allowed  to  stand  over  night.  It  was  then  filtered,  and  a  part 
of  the  clear  filtrate  first  obtained  was  precipitated  by  neutralization  with  0.2 
per  cent  hydrochloric  acid.  This  precipitate  was  washed  with  water,  alco- 
hol, absolute  alcohol,  and  ether,  yielding  preparation  93.  A  part  of  this 
preparation,  93,  was  redissolved  in  0.2  per  cent  potassium-hydroxide  water 
and  found  to  contain  a  considerable  amount  of  substance  which  had  become 
insoluble  in  consequence  of  drying.  This  insoluble  portion  was  filtered  off, 
washed  with  water,  alcohol,  and  ether,  and  gave  preparation  94.  The  filtrate 
from  this  substance  was  precipitated  with  0.2  per  cent  hydrochloric  acid, 
and  the  precipitate  filtered  off  and  washed  with  water,  alcohol,  and  ether. 
Through  an  accident  this  preparation  dried  on  the  filter  and  could  not  be 
removed  from  the  paper.  It  was  then  again  dissolved  in  dilute  potassium- 
hydroxide  water  and  treated  exactly  as  before,  yielding  preparation  95. 
The  following  analyses  show  the  composition  of  these  three  preparations  : 

Preparations  93,  94,  and  95. 


I. 

II. 

Average. 

Ash-free. 

Preparation  93  : 
Carbon  

P.ct. 
SI.SQ 

P.ct. 

P.ct. 
51.  5Q 

P.ct. 
S2.32 

Hydrogen  .... 

6.72 

6.72 

6.82 

Nitrogen  

17.34 

17.  3Q 

17.37 

17.61 

Sulphur  

2^.  21? 

Oxygen  
Ash  

1  

1.40 

IOO.OO 

Preparation  94  : 
Carbon  

5O.7Q 

50.  7Q 

152.87 

Hydrogen  

6.62 

6.62 

6.88 

Nitrogen  

16.38 

16.38 

16.38 

1  7.  OS 

Sulphur  

2^.2O 

Oxygen  
Ash  

j   

3.Q4 

VQ4 

IOO.OO 

Preparation  95  : 
Carbon  

ci  .  so 

ci  .-27 

CT  44 

52.62 

Hydrogen  

6.7O 

6.S7 

6.64 

6.80 

Nitrogen  

16.70 

16.70 

17.08 

Sulphur  

i  

2'j    TO 

Oxygen  
Ash  

2.2$ 

IOO.OO 

86 


THE;  PROTEINS  OE  THE  WHEAT  KERNEL. 


Another  lot  of  gluten  made  from  TOGO  grams  of  "straight  flour"  was 
treated  in  exactly  the  same  way  as  that  last  described.  This  gluten  was 
thoroughly  extracted  with  alcohol  of  0.90  sp.  gr.  and  the  residue  dissolved 
in  about  500  cc.  of  0.2  per  cent  potassium-hydroxide  water.  After  standing 
over  night,  a  very  turbid  liquid  was  decanted  from  the  sediment  which  had 
formed  and  treated  with  very  dilute  acetic  acid  added  to  slightly  acid  reac- 
tion. The  precipitate  produced  was  washed  with  water,  alcohol,  and  ether 
and  dissolved  again  in  0.2  per  cent  potassium-hydroxide  water.  The  result- 
ing solution,  filtered  perfectly  clear,  was  precipitated  with  0.2  per  cent 
hydrochloric  acid,  washed  by  decantation  with  water,  then  with  dilute 
alcohol  increased  gradually  in  strength  up  to  absolute  alcohol,  and  finally 
with  ether.  When  dried  over  sulphuric  acid,  a  pure  white,  light  mass  was 
obtained.  This  preparation,  96,  was  analyzed  with  the  results  shown  in  the 
table  below. 

Again,  gluten  was  prepared  in  the  usual  manner  and  extracted  with  alcohol 
until  everything  soluble  in  that  reagent  was  removed.  The  residue  was  then 
dissolved  in  0.2  per  cent  potassium-hydroxide  water  and,  in  order  to  carry 
the  operation  to  an  end  as  rapidly  as  possible,  the  solution  was  at  once 
thrown  into  a  filter.  As  soon  as  the  filtrate  ceased  to  pass  through  turbid 
it  was  returned  to  the  funnel  and  the  filtration  continued  in  an  ice-chest. 
A  considerable  portion  of  the  solution  was  obtained  perfectly  clear  after  20 
hours.  This  was  then  precipitated  with  o.  2  per  cent  hydrochloric  acid  and 
treated  in  the  usual  manner,  giving  preparation  97. 

Preparations  96  and  97. 


Preparation  96. 

Preparation  97. 

I. 

II. 

Average. 

Ash-free. 

I. 

Ash-free. 

Carbon  
Hydrogen  .  . 
Nitrogea  
Sulphur  .... 

P.  ct. 
52.07 
6.71 
17.23 
i  07 

P.  ct. 
52-23 
6.88 
17.42 

P.ct. 
52.15 
6.80 

17-33 
1.07 

P.  Ct. 

52.54 
6.85 
17.46 
1.07 
22.08 

P.  ct. 
52.07 
6.77 

17-49 
1.24 

P.  ct. 

52.38 
6.81 

17-59 
1.24 
21.98 

Ash  

0.74 

0.76 

o.75 

0.61 

100.00 

100.00 

In  order  to  determine  whether  a  loss  of  nitrogen  occurred  through  pro- 
longed contact  with  the  alkaline  solution,  the  following  experiment  was  tried, 
in  which  the  conditions  under  which  preparation  97  was  made  were  repeated 
and  preparation  98  obtained.  The  rest  of  the  alkaline  solution  was  kept  in 
the  ice-box  for  3  days  longer,  during  which  time  the  ice  melted  and  the 


EXPERIMENTAL. 


temperature  rose  to  20°.  The  preparation,  99,  obtained  by  neutralizing  the 
solution  with  hydrochloric  acid  contained  the  same  amount  of  nitrogen  as 
preparation  98,  from  which  it  is  evident  that  prolonged  solution  in  dilute 
potassium  hydroxide  solution  caused  no  loss  of  nitrogen. 

Preparations  98,  99,  and  100. 


Preparation  98. 

Preparation  99. 

Preparation  100. 

I. 

Ash-  free. 

I. 

Ash-free. 

I. 

II. 

Average. 

Ash-free. 

Nitrogen  .  .  . 
Ash  

P.  ct. 
17.32 

1.2^ 

P.ct. 
17-53 

P.ct. 
I7-50 
o.  16 

P.ct. 
17.53 

P.ct. 

17.04 

1.22 

P.ct. 
16.93 

P.ct. 
16.99 

P.ct. 
17.20 

Another  preparation  of  this  substance  (preparation  100)  was  made  by 
completely  extracting  200  grams  of  "patent  flour"  (from  spring  wheat) 
with  large  quantities  of  alcohol  of  0.90  sp.  gr.  The  flour  was  then  washed 
with  absolute  alcohol  and  air-dried.  The  dry  material  was  then  powdered 
and  made  into  a  dough  with  distilled  water  which  had  considerable  coher- 
ence, showing  that  the  protein  insoluble  in  alcohol  played  an  important  part 
in  its  formation.  This  dough  was  then  washed  on  a  fine  hair-sieve  under  a 
stream  of  running  water,  but  no  coherent  gluten  resulted.  The  washings 
were  then  allowed  to  settle,  and  the  sediment,  after  decanting  the  solution, 
was  treated  with  0.2  per  cent  potassium-hydroxide  water.  The  solution  so 
obtained,  after  standing  over  night,  was  decanted  from  the  sediment  and 
precipitated  with  o.  2  per  cent  hydrochloric  acid  and  the  separated  protein 
allowed  to  settle.  The  solution  was  then  decanted,  the  precipitate  dissolved 
in  0.2  per  cent  potassium-hydroxide  water,  and  filtered  clear  in  the  ice-box. 
The  filtered  solution  was  then  precipitated  and  the  separated  substance 
treated  in  the  usual  manner.  This  preparation,  100,  contained  nitrogen,  as 
shown  in  the  above  table. 

In  order  to  learn  whether  any  change  in  the  protein  occurred  in  conse- 
quence of  contact  with  aqueous  solutions  before  extracting  with  potassium- 
hydroxide  water,  the  following  experiment  was  made  : 

1000  grams  of  "straight  flour"  from  spring  wheat  were  repeatedly  ex- 
tracted with  alcohol  of  0.90  sp.  gr.,  and  after  removing  everything  soluble 
in  that  liquid  the  alcohol  was  squeezed  out  as  completely  as  possible  in  a 
screw-press  and  the  residue  extracted  with  o.  2  per  cent  potassium-hydroxide 
water.  It  was  found  impossible  to  separate  the  solution  from  the  undis- 
solved  portion  of  the  meal,  either  by  filtration  or  subsidence,  on  account  of 
the  presence  of  gummy  matter.  An  equal  volume  of  alcohol  of  sp.  gr.  0.820 


THE    PROTEINS   OF    THE    WHEAT    KERNEL. 


was  therefore  added,  and  on  long  standing  the  insoluble  substance  gradually 
settled,  leaving  a  comparatively  clear,  yellow  solution.  This  was  then 
siphoned  off  and  filtered.  On  account  of  its  gummy  character  it  filtered 
very  slowly.  The  clear  solution  finally  obtained  was  then  precipitated  with 
0.2  per  cent  hydrochloric  acid,  the  precipitate  filtered  off  and  dissolved  in 
0.2  per  cent  potassium-hydroxide  water.  The  solution  so  obtained  was 
filtered  clear  and  precipitated  with  0.2  per  cent  hydrochloric  acid,  the  result- 
ing precipitate  washed  by  decantation  with  water,  alcohol — at  first  dilute, 
then  gradually  increased  to  absolute — and  finally  with  ether.  Preparation 
101  was  thus  obtained,  having  the  composition  shown  in  the  following  table  : 

Preparations  101  and  102. 


Preparation  101. 

Preparation  102. 

I. 

Ash-free. 

I. 

II. 

Average. 

Ash-free. 

Carbon  
Hydrogen  .  . 
Nitrogen  .  .  . 
Sulphur  . 
Oxygen  
Ash 

P.  ct. 

52.14 
6.91 

17-54 

O.IO 

P.  ct. 

52.19 
6.92 

17.56 
23-33 

P.  ct. 

51-59 
6.85 
17.21 
f   0.89 

P.  ct. 
51.67 
6.87 
17.32 

P.ct. 

51-63 
6.86 
17.27 
0.89 

P.  ct. 
52.19 
6-93 
17-45 
0.90 

23-43 

I.  O7 

IOO.OO 

IOO.OO 

This  analysis  shows  that  the  protein  extracted  by  potassium  hydroxide 
water  from  the  flour  which  has  not  been  in  contact  with  water  has  the 
same  composition  as  that  obtained  in  the  other  experiments. 

PROTEIN  EXTRACTED  BY  DILUTE  ALKALINE  SOLUTIONS  AFTER  COMPLETE  EXTRACTION 
WITH  DILUTE  ALCOHOL  OF  GLUTEN  FROM  WHOLE-WHEAT  FLOUR. 

looo  grams  of  flour  made  by  grinding  the  entire  kernel  of  spring  wheat 
were  made  into  a  dough,  washed  with  water  till  free  from  starch,  and  the 
gluten  obtained  extracted  thoroughly  with  dilute  alcohol.  The  residue  was 
then  dissolved  in  0.2  per  cent  potassium-hydroxide  water,  and  after  the 
resulting  solution  had  stood  for  some  time  it  was  decanted  from  the  sedi- 
ment and  precipitated  by  0.2  percent  hydrochloric  acid.  The  precipitate 
was  washed  by  decantation  with  water,  thoroughly  extracted  with  dilute 
alcohol  and  then  with  absolute  alcohol,  and  finally  with  ether,  and  then 
redissolved  in  0.2  per  cent  potassium-hydroxide  water. 

The  solution,  after  filtering  perfectly  clear,  was  precipitated  and  the  pre- 
cipitate treated  in  the  same  manner  as  all  the  preceding  preparations.  When 


EXPERIMENTAL. 


89 


analyzed,  preparation  102  was  found  to  have  the  composition  shown  in  the 
table  on  page  88. 

The  filtrate  from  this  preparation,  as  well  as  all  the  others  previously 
described,  contained  a  small  amount  of  protein  matter.  This  was  then 
treated  with  a  solution  of  copper  sulphate  and  the  small  precipitate  thereby 
produced  filtered  off,  washed  with  water,  alcohol,  and  ether  (preparation 
103),  and  found  to  contain  the  following  amount  of  nitrogen  : 

Preparation  103. 


I. 

Ash-free. 

Nitrogen  
Ash        

P.  Ct. 

13.28 
23.88 

P.ct. 
17-45 

In  the  same  way  as  102  a  preparation  of  this  protein  was  made  from 
flour  from  the  entire  kernel  of  winter  wheat.  The  composition  of  this 
preparation  is  shown  by  the  figures  in  the  following  table  for  preparation 
104. 

Preparations  104  and  105. 


Preparation  104. 

Preparation  105. 

I. 

II. 

Average. 

Ash-free. 

I. 

Ash-free. 

Carbon  ...    . 
Hydrogen    . 
Nitrogen.  .    . 
Sulphur  .  .    . 
Oxygen.  .  .   . 
Ash  

P.  Ct. 

5I.7I 
6.79 
17.32 

)... 

P.ct. 

P.ct. 
5L7r 
6.79 

17.38 

P.ct. 

52.03 
6.83 
17.48 

23.66 

P.ct. 
51.17 
6.70 

17-35 

P.ct. 

52.44 
6.86 
17.81 

22.89 

17.44 

/ 
0.62 

0.62 

2.62 

IOO.OO 

100.00 

A  second  portion  of  the  same  solution  from  which  preparation  104  had 
been  precipitated  was  obtained  2  days  later.  This  was  then  treated  in 
the  same  way  and  yielded  preparation  105,  which  was  found  to  have  the 
composition  shown  in  the  table  above. 

If  preparations  89  and  92  are  omitted  on  account  of  being  obtained  from 
unfiltered  solutions,  94  as  an  altered  and  insoluble  product,  and  95  as  having 
been  subjected  to  an  exceedingly  prolonged  action  of  an  alkaline  solution, 
then  will  be  had,  in  table  18,  the  analyses  which,  in  the  opinion  of  the 
writer,  most  nearly  represent  the  true  composition  of  this  protein. 


9O  THE    PROTEINS    OF   THE    WHEAT    KERNEL. 

TABLE  18. — Protein  of  the  wheat  kernel  soluble  only  in  dilute  acids  and  alkalis. 


90. 

91- 

93- 

96. 

97- 

98 

99- 

Carbon  

P.ct. 

52.  2Q 

P.ct. 

P.ct. 

52.^2 

P.ct. 

52.54 

P.ct. 

52.38 

P.ct. 

P.ct. 

Hydrogen  

6.6l 

6.82 

6.85 

6.81 

Nitrogen  

17.4-1 

I7.T.T. 

I7.6l 

17.46 

17.  5Q 

17.5^ 

17.5^ 

Sulphur 

.04 

I 

f       1.07 

1.24 

Oxvsren.  . 

22.75 

}      23.25 

\    22.08 

2I.Q8 

IOO.OO 

IOO.OO 

IOO.OO 

IOO.OO 

IOO. 

101. 

102. 

103. 

104. 

105. 

Average. 

Carbon  

P.ct. 

P.ct. 

52.  IQ 

P.ct. 

52   IQ 

P.ct. 

P.ct. 

$2  O7 

P.ct. 

52  44 

P.ct. 

52   1A 

Hydrogen  .... 

6.Q2 

6.0^ 

6.81 

6  86 

6  81 

Nitrogen  

17.20 

17.^6 

17.45 

17  45 

17  48 

17  81 

17  4Q 

Sulphur  

1 

/      I  08 

r  23.33 

23-43 

23.66 

22.89 

IOO.OO 

IOO.OO 

IOO  OO 

IOO  OO 

IOO  OO 

There  can  be  no  doubt  that  the  earlier  analyses  of  this  protein,  with  the 
exception  of  those  made  by  Ritthausen  and  Chittenden  &  Smith,  are  incor- 
rect, for  the  substance  analyzed  contained  a  large  part  of  the  impurities  of 
the  gluten,  since  after  extraction  with  alcohol  the  residue  was  not  dissolved 
in  any  solvent,  and  the  starch,  bran,  etc.,  contained  in  it  filtered  off.  Ritt- 
hausen's  figures  are  the  average  of  two  analyses  of  preparations  obtained 
from  solutions  in  o.  2  per  cent  potassium-hydroxide  water  which  had  been 
filtered,  one  perfectly  clear,  giving  a  product  containing  17.21  per  cent  N, 
the  other  very  nearly  clear  and  containing  17.08  per  cent  N. 

It  is  evident  that  the  alkaline  solution  of  the  gluten  that  has  previously 
been  extracted  with  cold  dilute  alcohol  contains  something  which  requires 
filtration  through  very  dense  paper  to  effect  its  separation.  Wheat  gluten 
contains  a  considerable  amount  of  phytocholesterin  and  lecithin,  together  with 
fat,  and  it  is  probable  that  all  these  bodies  are  held  suspended  in  the  solution 
of  the  dissolved  protein  as  an  emulsion,  and  can  only  be  removed  by  pre- 
cipitating the  first  solution  in  potassium-hydroxide  water,  extracting  the 
precipitate  with  alcohol  and  ether,  redissolving,  and  filtering  the  resulting 
solution  perfectly  clear.  The  first  alkaline  solution  before  precipitating  and 
extracting  with  alcohol  and  ether  can  not  be  filtered,  as  it  either  passes 
through  turbid  or  does  not  run  through  at  all. 

On  the  ground  of  priority  and  the  fact  that  the  relations  to  animal  proteins 
which  gave  rise  to  the  various  names  subsequently  applied  to  this  body  have 


EXPERIMENTAL.  91 

been  proved  to  have  no  foundation,  it  would  be  desirable  to  return  to  Taddei's 
original  name  and  in  future  call  this  protein  zymon.  Unfortunately  this 
name  is  derived  from  the  Greek  word  C^,  a  ferment,  and,  as  the  results  of 
this  investigation  show  that  the  supposed  ferment-changes  do  not  occur  in 
the  formation  of  gluten,  this  name  is  undesirable.  As  this  protein  is  espe- 
cially characteristic  of  gluten,  it  seems  appropriate  to  call  it  glutenin,  a  name 
suggested  by  Prof.  S.  W.  Johnson. 

HYDROLYSIS  OF  GLUTENIN. 

The  large  quantity  of  glutenin  which  was  required  for  the  quantitative 
determination  of  its  decomposition  products  was  prepared  from  the  residue  of 
the  wheat  gluten  after  extracting  the  gliadin  with  alcohol.  This  residue  was 
dried  at  room  temperature  and  then  ground  to  a  powder,  which  was  extracted 
first  with  absolute  alcohol  and  then  with  ether  as  long  as  either  solvent  re- 
moved anything  from  it.  The  alcohol  was  then  removed  at  room  temperature 
and  the  residual  powder  treated  with  just  enough  0.2  per  cent  solution  of 
potassium  hydroxide  to  dissolve  it.  The  resulting  turbid  solution  was  then 
filtered  perfectly  clear  and  neutralized  with  very  dilute  hydrochloric  acid. 
The  precipitate  produced  was  extracted  with  70  per  cent  alcohol  as  long  as 
any  gliadin  was  removed,  then  thoroughly  dehydrated  with  absolute  alcohol, 
and  dried  over  sulphuric  acid. 

940  grams,  equal  to  839.32  grams  of  glutenin,  ash  and  water  free,  were 
hydrolyzed  by  heating  for  several  hours  on  a  water-bath  with  a  mixture  of 
950  cc.  of  concentrated  hydrochloric  acid  and  950  cc.  of  water.  After  standing 
over  night,  the  solution  was  boiled  on  an  oil-bath  for  9  hours,  and  then 
saturated  with  hydrochloric  acid  gas.  By  the  same  treatment  as  that  applied 
to  gliadin  (p.  71)  202.73  grams  glutaminic  acid  hydrochloride,  equal  to 
162.40  grams  of  the  free  acid,  were  obtained.  Recrystallized  once  from 
concentrated  hydrochloric  acid,  this  melted  at  198°. 

Chlorine:  0.5386  gram  substance  gave  0.4211  gram  AgCl. 

Nitrogen:  0.5911  gram  substance  gave  NH3  =  4.53cc.  HC1  (i  cc.  HCl  =  o.oi  gram  N)_ 

Calculated  for  C5H10Oi  NCI,  Cl  19.35,  N  7.65  p.  ct.  ;  found,  Cl  19.33,  N  7.66  p.  ct. 

The  united  filtrates  and  washings  were  concentrated  to  a  sirup  on  a  water- 
bath  under  reduced  pressure  and  the  hydrochlorides  of  the  amino-acids 
esterified  three  times,  as  in  the  case  of  gliadin.  The  hydrochlorides  of  the 
esters  were  neutralized  and  the  free  esters  shaken  out  with  ether,  as  for 
gliadin.  After  drying  the  ether  solution  of  the  esters  with  potassium  car- 
bonate, it  was  kept  2  days  over  anhydrous  sodium  sulphate.  The  ether 
was  then  removed  by  distillation  from  a  water-bath  at  atmospheric  pressure 
and  the  esters  distilled,  with  the  results  shown  in  the  table  on  page  92  for 
distillation  A.  The  undistilled  residue  weighed  211.5  grams. 


THE    PROTEINS   OF   THE    WHEAT    KERNEL. 
Distillations  A  and  B. 


Fraction. 

Distillation  A. 

Fraction. 

Distillation  B. 

Temper- 
ature of 
bath 
up  to  — 

Pressure. 

Weight. 

Temper- 
ature of 
bath 
up  to  — 

Pressure, 

Weight. 

I  

o 

65 

IOO 
IOO 

no 

155 

20O 

ntm. 
I2.O 
12.0 

4.0 
1-5 
1.5 
0.8 

Grams. 

61.00 

43-74 
45-94 

33-01 

43-32 
32-56 

259-57 

I  

o 

65 

88 

IOO 

1  20 
180 

mm. 
12.0 
I2.O 
IO.O 

0.8 
0.8 

Grams. 
28.74 
24.27 
19.18 
23-23 
23.92 

II  

II  

TTT  fa.., 

TTT      fa.. 

mu... 

mU.  .::::. 

IV  

IV  

V  

119-34 

The  residue,  from  which  the  esters  had  been  removed  by  ether,  was  sub- 
jected to  two  or  more  esterifications,  as  in  the  case  of  gliadin,  and  the  esters 
distilled.  (See  distillation  B  in  the  table  above.)  The  undistilled  residue 
weighed  93  grams. 

The  different  fractions  from  the  two  distillations  were  worked  up  as  follows: 


Fraction. 

Temper- 
ature of 
bath 

Pressure. 

Weight. 

up  to  — 

T     f  A 

0 

65 

mm. 
12 

Grams. 
6l.OO 

MB.:::::::: 

65 

12 

28.74 

This  was  saponified  at  once  by  evaporating  to  a  sirup  with  concentrated 
hydrochloric  acid  and  the  residue  dissolved  in  alcohol  and  esterified  with 
dry  hydrochloric  acid  gas.  The  glycocoll  ester  hydrochloride  which  sepa- 
rated weighed  6.68  grams.  Recrystallized  from  alcohol,  this  melted  at  144°. 

Carbon  and  hydrogen:  0.3179  gram  substance  gave  0.3984  gram  CO2  and  0.2151  gram 

H2O. 
Calculated  for  C4H10OaNCl,  C  34.39,  H  7.23  p.  ct.;  found,  C  34.18,  H  7.52  p.  ct. 

The  filtrate  from  the  glycocoll  ester  hydrochloride  was  saponified  by 
evaporating  on  the  water-bath  with  hydrochloric  acid,  the  latter  removed 
with  silver  sulphate,  and  the  solution  freed  from  sulphuric  acid  with  an 


EXPERIMENTAL. 


93 


equivalent  quantity  of  barium  hydroxide.     By  fractional  crystallization  this 
solution  yielded  11.83  grams  of  alanine,  which  melted  at  about  290°. 

Carbon  and  hydrogen:    0.2701  gram  substance,  dried  at  110°,  gave  0.4001  gram  CO2 

and  0.1946  gram  H2O. 
Nitrogen:  0.3460  gram  substance,  dried  at    110°,  gave   NH3  =  5.44  cc.   HC1   (i  cc. 

HCl  =  o.oi  gram  N). 
Calculated  for  C3H7O2N,  C  40.40,  H  7.93,  N  15.75  p.  ct.;  found,  C  40.40,  H  8.01,  N 

15-73  P-  ct. 


Temper- 

Fraction. 

ature  of 
bath 

Pressure. 

Weight. 

up  to  — 

o 

mm. 

Grams. 

-T     f   A.. 

IOO 

12 

43.74 

11  \B... 

88 

IO 

24.27 

The  united  esters  were  saponified  by  boiling  with  10  parts  of  water  for  5 
hours,  when  their  solution  reacted  neutral  to  litmus.  This  solution  was 
evaporated  to  dry  ness  under  reduced  pressure  and  boiled  up  with  absolute 
alcohol,  whereby  0.5  gram  substance  was  dissolved.  By  repeated  fractional 
crystallization  of  the  substance  insoluble  in  alcohol,  3.5  grams  leucine  were 
obtained.  The  isolated  leucine  decomposed  at  about  298°. 

Carbon  and  hydrogen:  0.2408  gram  substance,  dried  at  110°,  gave  0.4852  gram  CO2  and 

0.2169  gram  H2O. 
Calculated  for  C6H13O2N,  C  54.89,  H  10.01  p.  ct. ;  found,  C  54.95,  H  10.01  p.  ct. 

After  adding  to  the  filtrate  from  this  leucine  the  most  soluble  portion  of 
fraction  III,  and  further  fractioning,  25.76  grams  of  alanine  and  3. 84  grams 
of  glycocoll  were  obtained.  The  latter  was  isolated  as  the  hydrochloride 
of  the  ester,  which  melted  at  144°.  The  alanine  was  racemized  and  the 
a-naphthyl-hydantoic  acid  prepared  according  to  the  directions  of  Neuberg  & 
Manasse.1  When  recrystallized  from  dilute  alcohol,  this  melted  at  197°. 

Carbon  and  hydrogen:    0.2527  gram  substance,  dried  at  90°,  gave  0.5998  gram  CO2 

and  0.1280  gram  H2O. 
Nitrogen:  0.3515    gram    substance,  dried  at  90°,  gave   NH3  =  3.74  cc.  HC1  (i  cc. 

HC1  =  o.oi  gram  N). 
Calculated  for  CUHUO3N2,    C  65.06,  H  5.48,  N  10.87  p.  ct.;  found,  C  64.74,  H  5.61, 

N  10.61  p.  ct. 


1  Neuberg  &  Manasse,  Berichte  der  deutschen  chemischen  Gesellschaft,  1905,  xxxvm, 
P-  2359- 


94 


THE;  PROTEINS  OF  THE  WHEAT  KERNEL. 


Fraction. 

Temper- 
ature of 
bath 

Pressure. 

Weight. 

up  to  — 

rr    f    A.. 

0 

no 

mm. 

T    C 

Grams. 
78  QS 

m{  £::::::: 

1  2O 

0.8 

4.2  41 

These  esters  were  saponified  by  boiling  with  6  parts  of  water  for  5  hours, 
when  their  solution  reacted  neutral  to  litmus.  After  evaporating  to  dryness 
under  reduced  pressure,  the  dried  residue  was  extracted  with  boiling  absolute 
alcohol.  The  part  insoluble  in  alcohol,  by  a  systematic  fractional  crystal- 
lization, gave  32.42  grams  of  leucine,  which  decomposed  at  about  298°. 

Carbon  and  hydrogen:    0.4255  gram  substance,  dried  at  no0,  gave  0.8535  gram  CO2 

and  0.3926  gram  H2O. 

Nitrogen:  0.1488  gram  substance  gave  NH3=  i.6occ.  HC1  (i  cc.  HCl  =  o.oi  gram  N). 
Calculated  for  C6H13O2N,  C  54.89,  H  10.01,  N  10.70  p.  ct;  found,  C  54.71,  H  10.25. 

N  10.77  P-  ct. 

The  filtrate  from  this  leucine  yielded  2  grams  of  substance,  which  had  the 
composition  and  properties  of  amino-valerianic  acid. 

Carbon  and  hydrogen:   0.2216  gram  substance  gave  0.4144  gram  CO2  and  0.1967  gram 

H20. 
Calculated  for  C5HUO2N,  C  51.22,  H  9.48  p.  ct.;  found,  C 51.00,  H  9.86  p.  ct. 

Specific  rotation. — Dissolved  in  20  per  cent  hydrochloric  acid, 

00  ^-=+25.63° 

A  similar  preparation  from  gliadin  gave +25. 79°.  B.  Fischer  &  Dor- 
pinghaus1  found  +25.9°  for  a  preparation  from  horn,  and  B.  Schulze  & 
Winterstein2  found  +28.2°  and  +27.9°  for  a  preparation  from  lupine 
seedlings. 

The  solution  used  for  determining  the  specific  rotation  was  freed  from 
hydrochloric  acid  with  silver  sulphate,  and  the  amino-acids  racemized  by 
heating  with  an  excess  of  barium  hydroxide  in  an  autoclave  at  175°  for  19 
hours.  After  removing  the  barium  quantitatively  with  sulphuric  acid,  the 
substance  was  coupled  with  a-naphthylisocyanate.  The  hydantoic  acid 
melted  constantly,  on  repeated  recrystallization  from  dilute  alcohol,  at  183°  to 

Fischer,  E.,  &  Dorpinghaus,  Zeitschrift  fur  physiologische  Chemie,  1902,  xxxvi, 
p.  462. 
2 Schulze,  B.,  &  Winterstein,  ibid.,  1902,  xxxv,  p.  300. 


EXPERIMENTAL.  95 

184°.     Heated  side  by  side  with  the  corresponding  substance  obtained  from 
gliadin,  the  hydantoic  acid  from  glutenin  melted  at  2°  higher. 

Carbon  and  hydrogen:   0.3277  gram  substance,  dried  at  80°,  gave  0.8067  gram  CO2  and 

0.1891  gram  H.2O. 
Calculated  for  C16H13O3N.2)  C  67.07,  H  6.35  p.  ct;  found,  C  67.14,  H  6.42  p.  ct. 

From  the  filtrate  from  the  amino-valerianic  acid  there  was  further  obtained 
1.41  grams  of  alanine.  The  alcoholic  solution  which  contained  the  proline 
was  evaporated  to  dryness  under  reduced  pressure  and  the  dried  residue 
again  treated  with  boiling  absolute  alcohol.  Even  after  several  repetitions 
of  this  process  no  substance  insoluble  in  alcohol  could  be  obtained.  The 
alcohol-soluble  substance,  when  dried,  weighed  35.54  grams.  A  copper  salt 
was  prepared  from  this  in  the  usual  manner,  and  its  solution  evaporated  to 
dryness  under  reduced  pressure.  The  dried  residue  was  extracted  with  boil- 
ing absolute  alcohol  in  order  to  remove  the  /-proline  copper.  The  residue 
insoluble  in  alcohol,  when  recrystallized  from  water,  gave  15.53  grams  of 
racemic  proline  copper,  equivalent  to  10.9  grams  of  proline. 

Water :  0.3618  gram  substance  lost  at  110°  0.0399  gram  of  H2O. 
Copper :  0.3176  gram  substance,  dried  at  110°,  gave  0.0856  gram  CuO. 
Calculated  for  C10H16O4N2  Cu  •  2H.2O,  H2O  ir.oo  p.  ct.  ;  found,  HaO  11.03  P-  ct. 
Calculated  for  C10H16O4N2  Cu,  Cu  21.79  P-  ct.  ;  found,  Cu  21.54  P-  ct. 

The  alcoholic  solution  of  the  /-proline  copper  salt  was  evaporated  to  dry- 
ness,  the  copper  removed,  and  the  proline  identified  as  the  phenylhydantoin 
which  was  prepared  according  to  the  directions  of  Fischer.1  The  substance 
thus  prepared  was  at  once  pure  and  melted  at  143°. 

Carbon  and  hydrogen:  0.2822  gram  substance,  dried  in  vacuo  over  H2SO4,  gave  0.6866 

gram  CO2  and  0.1494  gram  H2O. 

Nitrogen:  0.1025  gram  substance  gave  NH3  =  1.33  cc.  HC1  ( i  cc.  HC1  =  o.oi  gram  N). 
Calculated  for  C12H12O2N2,  C  66.60,  H  5.61,  N  12.99  p.  ct.  ;   found,  C  66.36,  H  5.88, 

N  12.98  p.  ct. 

f  Fraction  IV,  A.     Temperature  of  bath,  up  to  155°.  ) 
(Pressure,  1.5  mm.     Weight,  43.32  grams.  j 

This  fraction  was  shaken  out  with  ether  in  the  usual  way  and  the  ether 
allowed  to  evaporate  spontaneously.  No  evidence  of  the  presence  of  phenyl- 
alauine  was  obtained.  The  residue  of  ester  was  saponified  with  concentrated 
hydrochloric  acid  and  the  hydrochloride  decomposed  with  ammonia.  The 
free  acid  crystallized  in  the  characteristic  form  of  leucine  and  decomposed  at 
298°.  There  were  obtained  13.99  grams  of  leucine. 

Carbon  and  hydrogen:  0.2906  gram  substance,  dried  at  110°,  gave  0.5825  gram  CO2 

and  0.2690  gram  H2O. 
Calculated  for  C6H13O2N,  C  54.89,  H  10.01  p.  ct.  ;  found,  C  54.67,  H  10.28  p.  ct. 

Fischer,  E.,  Zeitschrift  fiir  phystologische  Chemie,  1901,  xxxin,  p.  151. 


96 


THE    PROTEINS   OF   THE    WHEAT    KERNEL. 


The  aqueous  layer  was  saponified  by  heating  on  a  water-bath  with  an  excess 
of  barium  hydroxide  for  5  hours.  The  barium  aspartate,  which  separated  in 
considerable  quantity  on  standing,  was  united  with  that  obtained  from  frac- 
tion V  and  treated  as  will  be  described  later. 

From  the  filtrate  from  the  barium  aspartate  no  definite  substance  could  be 
obtained.  It  appeared  to  contain  serine,  but  none  could  be  isolated,  even  by 
the  use  of  /3-naphthalene-sulphone-chloride. 


Temper- 

Fraction. 

ature  of 
bath 

Pressure. 

Weight. 

up  to  — 

o 

mm. 

Grams. 

V,  A  

2OO 

0.8 

^2.56 

IV.  B  .  . 

1  80 

0.8 

2^.Q2 

These  esters  were  shaken  out  with  ether,  and  the  substance  extracted 
was  saponified  with  hydrochloric  acid.  The  hydrochloride  thus  obtained, 
which  weighed  20.21  grams,  equal  to  16.55  grams  free  phenylalanine,  was 
converted  into  the  free  acid  with  ammonia  and  then  into  the  copper  salt  by 
boiling  its  solution  with  copper  hydroxide.1 

Copper :  0.2099  gram  substance,  dried  at  110°,  gave  0.0425  gram  CuO. 

Nitrogen:  0.2283  gram  substance  gave  NH3  —  1.63  cc.  HC1  (i  cc.  HCl  =  o.oi  gram  N). 

Calculated  for  C18H20O4N2Cu,  Cu  16.23,  N  7.17  p.  ct.;  found,  Cu  16.18,  N  7.14  p.  ct. 

The  free  phenylalaniiie  isolated  from  this  copper  salt  melted  at  263°  to  265°. 

Nitrogen:  0.1085  gram  substance  gave  NH3  =  0.92  cc.  HC1  (i  cc.  HC1  =  o.oi  gram  N). 
Calculated  for  C9HUO2N,  N  8.50  p.  ct.;  found,  N  8.48  p.  ct. 

The  aqueous  layer  was  saponified  by  heating  for  5  hours  with  an  excess 
of  barium  hydroxide  on  the  water-bath.  The  barium  aspartate,  which  sepa- 
rated on  standing,  was  united  with  that  previously  obtained  from  fraction 
IV,  A,  decomposed  with  an  equivalent  amount  of  sulphuric  acid,  and  7.12 
grams  of  free  aspartic  acid  were  obtained  from  the  solution. 

Carbon  and  hydrogen:  0.3293  gram  substance,  dried  at  110°,  gave  0.4335  gram  CO2and 

0.1625  gram  H2O. 

Nitrogen:  0.2997  gram  substance  gave  NH3  =  3.i7  cc.  HC1  (i  cc.  HC1  =  0.01  gram  N). 
Calculated  for  C^O^N,  C  36.05,  H  5.31,  N  10.55  P-   ct.  ;  found,  C  35.90,   H  5.48, 

N  10.58  p.  ct. 

The  filtrate  from  barium  aspartate  was  freed  from  barium,  concentrated 
under  reduced  pressure,  and  saturated  with  hydrochloric  acid.  On  long 
standing  on  ice  a  trace  of  phenylalanine  hydrochloride  separated,  but  no 
glutaminic  acid  hydrochloride  could  be  obtained.  After  removing  the 


JCf.  Schulze  &  Winterstein,  Zeitschrift  f ur  physiologische  Chemie,  1902,  xxxv,  p.  210. 


EXPERIMENTAL.  97 

hydrochloric  acid  with  silver  sulphate,  the  solution  was  boiled  with  a  solu- 
tion of  copper  hydroxide  and  i.i  grams  of  copper  aspartate  were  isolated. 

Copper :  0.3991  gram  substance,  air-dried,  gave  0.1163  gram  CuO. 

Nitrogen :  0.2066  gram  substance  gave  NH,  =  1.06  cc.  HC1  (i  cc.  HC1  =0.01  gram  N). 
Calculated  for  C4H5O4N,  Cu  •  4^  HjO,1  Cu  23.06,  N  5.09  p.  ct.  ;  found,  Cu  23.27,  N  5.13 
p.  ct. 

The  filtrate  from  this  copper  aspartate,  when  freed  from  copper  by  hydro- 
gen sulphide  and  concentrated  to  small  volume,  yielded,  by  fractional  crys- 
tallization, 4.35  grams  of  nearly  pure  serine. 

Carbon  and  hydrogen:  0.2847  gram  substance  gave  0.3559  gram  CO,  and  0.1751  gram 

H2O. 
Calculated  for  C3H7O3N,  C  34.29,  H  6.67  p.  ct.  ;  found,  C  34.09,  H  6.83  p.  ct. 

This  substance  browned  at  about  2 13°  and  decomposed  to  a  brownish  mass 
at  about  243°. 

RESIDUE  FROM  DISTILLATION. 

The  residues  which  remained  after  distilling  off  the  esters  weighed  304.5 
grams.  These  were  dissolved  in  hot  alcohol,  and  from  their  united  solutions 
3.16  grams  of  substance  separated  on  cooling.  The  filtrate  from  this  sub- 
stance was  evaporated  under  reduced  pressure,  the  residue  dissolved  in  water 
and  saponified  by  heating  with  an  excess  of  barium  hydroxide  for  9  hours. 
After  removing  the  barium  quantitatively,  the  solution  was  concentrated  and 
saturated  with  hydrochloric  acid.  On  standing  for  some  time  on  ice,  38.25 
grams  of  glutaminic  acid  hydrochloride,  which  melted  at  198°,  were  ob- 
tained. This  was  equivalent  to  30.64  grams  of  free  glutaminic  acid,  and, 
with  that  previously  isolated,  made  a  total  of  193.04  grams,  or  23  per  cent 
of  the  glutenin.  The  glutaminic  acid  hydrochloride  was  decomposed  with 
an  equivalent  quantity  of  potassium  hydroxide,  and  the  free  glutaminic  acid 
was  recrystallized  from  water.  It  melted  at  202°  to  203°  with  effervescence. 

Carbon  and  hydrogen:  0.3504  gram  substance,  dried  at  110°,  gave  0.5275  gram  CO, 

and  0.2003  gram  H,O. 
Calculated  for  C5H9O4N,  C  40.82,  H  6.12  p.  ct.  ;  found,  C  41.06,  H  6.35  p.  ct. 

RESIDUE  AFTER  ESTERIFICATION. 

The  residue  which  remained  after  the  third  esterification  and  extraction 
of  the  esters  was  treated  in  the  same  way  as  the  corresponding  residue  from 
gliadin  (p.  77),  and  the  solution,  freed  from  all  mineral  salts  and  bases 
precipitable  by  phosphotungstic  acid,  was  concentrated  under  reduced  press- 
ure to  a  small  volume  and  then  left  for  some  time  over  sulphuric  acid. 

^itthausen,  Die  Eiweisskorper,  etc.,  Bonn,  1872,  p.  219. 
7 


98  THE  PROTEINS  OE  THE  WHEAT  KERNEL. 

After  removing  a  little  tyrosine  that  first  separated,  1.87  grams  of  seriiie 
crystallized  out,  which,  when  recrystallized  from  water,  browned  at  about 
210°  and  decomposed  at  240°. 

Carbon  and  hydrogen:   0.2308  gram  substance,  dried  at  110°,  gave  0.2894  gram  CO2  and 

o.  1407  gram  H2O. 
Calculated  for  C3HTO3N,  C  34.29,  H  6.67  p.  ct.;  found,  C  34.19,  H  6.77  p.  ct. 

This,  with  the  serine  obtained  from  fraction  V,  gives  a  total  of  6.22  grams 
of  serine  isolated.  The  mother-liquor  from  the  serine  contained  consider- 
able substance,  but  no  oxy-proline  or  other  definite  substance  could  be 
obtained  from  it. 

CYSTINE. 

300  grams  of  glutenin  were  hydrolyzed  in  the  way  described  for  gliadin 
(p.  80).  After  evaporating  at  low  pressure  to  a  sirup,  neutralizing  the 
remaining  excess  of  acid  with  sodium  hydroxide,  and  decolorizing  the  solu- 
tion with  bone-black,  a  considerable  quantity  of  tyrosine  separated  out, 
which,  on  examination,  was  found  to  contain  nearly  all  the  cystine  that 
could  be  detected  in  the  solution.  It  was  therefore  dissolved  in  5  per  cent 
sulphuric  acid  and  the  cystine  precipitated  with  mercuric  sulphate.  The 
mercury  precipitate  was  decomposed  with  hydrogen  sulphide,  the  solution 
concentrated  somewhat,  made  alkaline  with  ammonia  and  then  acid  with 
acetic  acid,  and  an  equal  volume  of  alcohol  added.  The  cystine,  which  sep- 
arated on  standing  in  characteristic  hexagonal  plates,  weighed  only  0.17 
gram.  No  more  could  be  obtained.  This  was  dissolved  in  ammonia  and 
reprecipitated  by  acetic  acid. 

Sulphur :  0.0897  gram  substance,  dried  at  110°,  gave  0.1730  gram  BaSO4. 
Calculated  for  CgHuC^NjS.,,  S  26.68  p.  ct.;  found,  S  26.53  P-  ct. 

Although  glutenin  contains  about  the  same  amount  of  sulphur  as  gliadin, 
the  amount  of  cystine  obtained  from  the  latter  under  similar  conditions  was 
very  much  greater.  It  would  seem  as  if  glutenin  in  fact  yields  less  cystine, 
though  the  uncertainties  attending  the  isolation  of  this  substance  will  not 
permit  of  a  positive  conclusion. 

TYROSINE. 

250  grams  of  glutenin  were  boiled  with  a  mixture  of  750  grams  sulphuric 
acid  and  1500  grams  of  water  for  12  hours.  The  solution  was  freed  from 
sulphuric  acid  by  an  equivalent  amount  of  barium  hydroxide,  and  after 
concentrating  to  800  cc.  allowed  to  stand  for  some  time.  A  considerable 
quantity  of  tyrosine  separated,  which  was  filtered  out,  the  filtrate  boiled 
with  barium  carbonate  in  order  to  expel  ammonia,  and  then  concentrated  to 
one-half  its  original  volume.  After  cooling,  the  residue  of  barium  carbonate 


EXPERIMENTAL.  99 

and  other  substances  which  had  separated  were  extracted  with  hot  dilute 
ammonia  and  the  filtered  extract  concentrated  and  cooled.  On  standing,  a 
little  more  tyrosine  separated,  which  was  added  to  that  first  obtained.  No 
more  tyrosine  could  be  isolated  from  the  solution  of  the  hydrolytic  decom- 
position products.  All  of  the  tyrosine  which  had  separated  was  dissolved 
in  5  per  cent  sulphuric  acid,  and  phosphotungstic  acid  was  added  to  the 
solution.  Only  a  small  precipitate  resulted.  After  removing  the  phospho- 
tungstic acid  with  barium  hydroxide,  the  solution  was  concentrated  strongly 
and  allowed  to  cool.  After  standing  for  some  time,  9.62  grams  of  tyrosine 
were  obtained,  which  is  equal  to  4.25  per  cent  of  the  glutenin. 

Carbon  and  hydrogen:  0.2922  gram  substance,  dried  at  110°,  gave  0.6370  gram  COj  and 

0.1634  gram  H2O. 
Calculated  for  CaHnOjN.  C  59.62,  H  6.13  p.  ct.;  found,  C  59.45,  H  6.21  p.  ct. 


Kutscher1  found  2.75  per  cent  of  tyrosine  in  "  gluten-  casein." 

HISTIDINE. 

Fifty  grams  of  glutenin,  equal  to  43.39  grams  dried  at  110°,  were  hydro- 
lyzed,  and  the  arginine  and  histidine  separated  in  the  same  way  as  that 
described  for  gliadin  (p.  81).  The  solution  containing  the  histidine  was 
made  up  to  500  cc. 

Nitrogen:  100  cc.  solution  gave  NH3  =  4.15  cc.  HC1  (i  cc.  HC1  =0.01  gram  N)  = 
0.0415  gram  N  =  0.2075  gram  in  500  cc.  =  0.7645  gram  histidine  =  1.76  p.  ct.  of 
the  glutenin. 

The  amount  of  histidine  in  the  remaining  solution  was  too  small  for 
identification. 

ARGININB. 

The  filtrate  from  the  histidine  precipitate  yielded  500  cc.  of  solution  con- 
taining the  arginine,  in  which  was  found  the  following  amount  of  nitrogen  : 

Nitrogen:  50  cc.  solution  gave  NHS  =  6.79  cc.  HC1  (i  cc.  HC1  =  o.oi  gram  N)  = 
0.0679  gram  N,  or  0.679  gram  in  500  cc.,  or  2.107  gram  arginine  =  4.72  p.  ct.  of  the 
glutenin. 

The  remaining  solution,  treated  as  Kossel  directs,  yielded  the  arginine  as 
carbonate.  This  was  converted  into  the  copper  salt,  which  gave  the  fol- 
lowing results  on  analysis  : 

Carbon  and  hydrogen:  0.2118  gram  substance,  air-dried,  lost  0.0210  gram  H2O  at  100°. 
Calculated  for  C12H,8O10Nj0  Cu  •  3  H2O,  H2O  9.15  p.  ct.  ;  found,  H2O  9.92  p.  ct. 
Copper  :  (I)  0.1858  gram  substance,  dried  at  100°,  gave  0.0275  gram  CuO  ;  (II)  0.1808 

gram  substance,  dried  at  100°,  gave  0.0267  gram  CuO. 
Calculated  for  C12H28O]0N10  Cu,  Cu  11.85  P-  ct.  ;  found,  Cu  (I)  11.84,  (II)  11.78  p.  ct. 

1  Kutscher,  Zeitschrift  fur  physiologische  Chemie,  1903,  xxxvi,  p.  114. 


IOO 


THE   PROTEINS  OF  THE   WHEAT    KERNEL. 


Kossel  &  Kutscher1  found  in  three  separate  determinations  of  arginine 
in  glutenin  4.50,  4.02,  and  4.54  per  cent.  They  base  their  determinations 
on  the  supposition  that  glutenin  contains  16.2  per  cent  nitrogen.  If  their 
results  are  recalculated  to  a  basis  of  17.5  per  cent  of  nitrogen  which  has  been 
found  in  this  protein,2  they  become  4.82,  4.52,  and  4.84  per  cent,  with 
which  4.72  per  cent  agrees  very  closely. 

LYSINE. 

The  filtrate  from  the  arginine  silver  was  treated  as  Kossel  directs,  and 
after  precipitating  the  lysine  with  phosphotungstic  acid  it  was  converted 
into  the  picrate,  and  2.33  grams,  equivalent  to  0.907  gram  of  free  lysine, 
were  obtained.  This  is  equal  to  1.92  per  cent  of  the  glutenin.  Kossel  & 
Kutscher  found  in  the  three  determinations  of  this  substance  which  they 
made  in  this  protein  1.9,  2.29,  and  2  per  cent,  or,  recalculating  to  a  basis  of 
17.5  per  cent  of  nitrogen  in  this  protein,  2.05,  2.15,  and  2.40  per  cent. 

Nitrogen:  0.1225  gram  substance,  dried  at  120°,  gave  23.1  cc.  moist  N2  at  760  mm. 

and  29°. 
Calculated  for  CgH^Nj  •  C6H3O7N3,  N  18.70  p.  ct.  ;  found,  N  18.76  p.  ct. 


The  results  of  this  hydrolysis  are  given  in  table  19. 

TABLE  19.  —  Glutenin. 


P.ct. 

Glycocoll 0.89 

Alanine 4.65 

Amino-valerianic  acid 0.24 

Leucine 5.95 

a-proline 4.23 

Phenylalanine 1.97 

Aspartic  acid 0.91 

Glutaminic  acid 23.42 

Serine 0.74 


P.  ct. 

Tyrosine 4.25 

Cystine 0.02 

I/ysine 1.92 

Histidine 1.76 

Arginine 4.72 

Ammonia 4.01 

Tryptophane present 

Total 59.66 


THE  AMOUNT  OF  THE  VARIOUS  PROTEINS   CONTAINED   IN  THE 
KERNEL  OF   WHEAT. 

looo  grams  of  fine  meal  obtained  by  freshly  grinding  the  entire  kernel  of 
spring  wheat  and  a  like  quantity  of  similar  flour  from  winter  wheat  were 
each  extracted  with  4000  cc.  of  10  per  cent  sodium- chloride  solution,  and 
2500  cc.  of  the  clear  extract  were  obtained  from  the  spring- wheat  flour  and 
2600  cc.  from  the  winter-wheat  flour.  As  100  cc.  of  solution  were  used  for 
every  25  grams  of  flour  taken,  the  amount  of  extract  obtained  was  approx- 
imately equal  to  that  from  625  grams  of  spring- wheat  flour  and  650  grams  of 
winter- wheat  flour.  These  extracts  were  then  dialyzed  until  all  the  chlo- 


1  Kossel  &  Kutscher,  Zeitschrift  fiir  physiologische  Chemie,  1900,  xxxi,  p.  165. 
*  Osborne  &  Voorhees,  American  Chemical  Journal,  1893,  xv,  392. 


EXPERIMENTAL.  IOI 

rides  were  removed,  which  required  5  days.  The  precipitated  globulin  was 
then  filtered  from  each,  washed  with  distilled  water,  alcohol,  absolute  alcohol, 
and  ether,  removed  from  the  filter,  and  dried  at  110°.  From  the  spring- 
wheat  extract  3.8398  grams  were  obtained,  equal  to  0.624  per  cent  of  the 
flour,  and  from  the  winter  wheat  3.9265  grams,  equal  to  0.625  per  cent. 

The  filtrates  from  the  globulin  were  then  heated  to  65°  in  a  water-bath, 
and  after  being  held  at  this  temperature  for  some  time  the  coagulum  was 
filtered  off  and  washed  with  hot  water,  alcohol,  and  ether,  removed  from 
the  paper,  and  dried  at  110°.  From  the  spring  wheat  1.9714  grams  were 
obtained,  being  0.315  per  cent  of  the  flour,  and  from  the  winter  wheat 
1.9614  grams,  equal  to  0.302  per  cent. 

The  solutions  filtered  from  each  of  these  coagula  were  next  heated  just 
to  boiling  and  the  resulting  coagulum  filtered  off,  washed  thoroughly,  and 
treated  as  the  preceding  preparations  had  been.  The  spring- wheat  extract 
thus  yielded  0.4743  gram,  equal  to  0.076  per  cent ;  the  winter-wheat  extract 
0.3680  gram,  equal  to  0.057  Per  cent. 

The  two  extracts  were  next  concentrated  by  boiling  down  over  a  lamp. 
They  remained  clear  at  first,  but  when  somewhat  concentrated  the  protein 
began  to  separate  as  a  skin  on  the  surface  of  the  solution.  When  reduced 
to  about  one-fourth  its  original  volume,  the  coagulum  was  filtered  off,  washed 
with  boiling  water,  alcohol,  and  ether,  and  dried  at  110°.  The  spring  wheat 
thus  yielded  0.8737  gram»  equal  to  0.139  per  cent ;  the  winter  wheat  0.8721 
gram,  equal  to  0.134  Per  cent.  The  filtrates  from  these  two  preparations 
were  evaporated  very  nearly  to  dryness  on  water-baths.  On  cooling,  much 
substance  separated,  which,  when  treated  with  hot  water,  dissolved  again. 
The  insoluble  coagulum  was  filtered  from  each,  washed,  and  dried  in  the 
usual  manner.  The  spring  wheat  gave  0.8149  gram  of  substance,  being 
0.130  per  cent  of  the  flour  ;  the  winter  wheat  0.5795  gram,  being  0.089  Per 
cent.  The  total  amount  of  protein  coagulating  on  concentration  was  there- 
fore 0.269  per  cent  for  the  spring  wheat  and  0.223  per  cent  for  the  winter 
wheat. 

The  filtrates  from  these  second  coagula  were  then  again  evaporated  to  a 
sirup,  and  as  no  more  insoluble  matter  separated  they  were  each  precipitated 
by  pouring  into  strong  alcohol.  Large  precipitates  resulted  in  each  case, 
which,  after  settling  and  decantation  of  the  alcohol,  were  dissolved  again  in 
a  little  water  and  precipitated  by  pouring  into  strong  alcohol.  Much  color- 
ing matter  and  sugar  was  held  in  solution,  as  proved  by  evaporation  of  the 
alcoholic  mother-liquors.  The  precipitates  were  then  thoroughly  dehydrated 
with  absolute  alcohol,  washed  with  ether,  and  dried  at  110°.  The  spring- 
wheat  extract  thus  yielded  6.9289  grams  of  substance,  the  winter  wheat 
8.7517  grams.  As  these  preparations  were  unquestionably  very  impure 


IO2 


THE   PROTEINS   OF   THE   WHEAT    KERNEL. 


and  no  practicable  method  of  purification  existed  which  could  be  carried  out 
without  great  loss  of  substance,  the  nitrogen  in  each  was  determined  and 
the  protein  calculated  by  multiplying  the  result  obtained  by  6.25.  The 
spring- wheat  preparation  was  thus  found  to  contain  3.07  per  cent  of  nitro- 
gen, equal  to  19.19  per  cent  of  protein,  and  the  winter-wheat  preparation 
5.15  per  cent  nitrogen,  equivalent  to  32.18  per  cent.  The  amount  of  pro- 
teose  and  peptone  thus  found  in  the  extract  from  the  spring  wheat  was 
1.3297  grams,  which  equals  0.213  per  cent  of  the  flour,  and  in  the  winter- 
wheat  extract  2.8063  grams,  equal  to  0.432  per  cent  of  the  flour. 

The  sodium  chloride  extract  contained,  therefore,  the  following  amounts 
of  protein : 


Spring 
wheat. 

Winter 
wheat. 

Globulin  

P.  ct. 

0.624 

P.  ct. 
o  62$ 

Albumin  '..... 

O.VJI 

O.^Q 

Coagulum  

0.260 

O.22T, 

Proteose  

O  21"* 

O.4A2 

1.497 

1.639 

The  remainder  of  the  protein  matter  of  the  seed  forms  the  gluten.  The 
proportion  of  gliadin  and  glutenin  in  this  gluten  was  determined  in  the  fol- 
lowing manner : 

200  grams  of  spring-wheat  flour  made  from  the  entire  seed  and  a  like 
quantity  of  a  similar  winter-wheat  flour  were  each  made  into  a  dough  and 
thoroughly  washed  with  water  as  long  as  starch  was  removed.  The  gluten 
thus  obtained,  after  freeing  from  loosely  adhering  moisture,  was  weighed 
and  exactly  one-half  dried  at  110°  to  constant  weight.  The  spring  wheat 
was  thus  found  to  yield  12.685  per  cent  dry  gluten,  the  winter-wheat  flour 
11.858  per  cent. 

The  other  half  of  the  gluten  was  cut  up  very  fine  and  extracted  as  thor- 
oughly as  possible  with  alcohol  of  0.90  sp.  gr.  The  alcoholic  extract  was 
then  evaporated  to  small  volume,  cooled,  and  the  solution  decanted  from  the 
precipitated  protein.  This  precipitate  was  then  extracted  with  ether  and 
dried  at  110°.  From  the  spring-wheat  gluten  4.3379  per  cent  and  from  the 
winter  wheat  4.2454  per  cent  of  alcohol-soluble  protein  were  obtained. 

The  residue  extracted  with  alcohol  was  then  dried  at  1 10°  and  weighed. 
The  spring- wheat  gluten  contained  7.80  per  cent  insoluble  in  alcohol,  the 
winter  wheat  7.504  per  cent,  reckoned  on  the  wheat.  Nitrogen  was  then 
determined  in  these  residues  dried  at  no°,  as  well  as  in  the  dried  gluten 


EXPERIMENTAL. 


103 


and  also  in  the  original  flours.  The  washings  of  the  glutens  were  collected 
in  jars  and  allowed  to  settle,  the  sediments  washed  with  water  and  with 
very  strong  alcohol  and  dried  and  weighed.  The  nitrogen  in  each  was  then 
determined.  The  results  of  these  determinations  and  deductions  drawn 
from  them  are  given  in  table  20. 

TABLE  20. — Amount  of  the  various  proteins  of  the  wheat  kernel. 


Spring 
wheat. 

Winter 
wheat. 

Total  nitrogen  in  the  flour  

P.  ct. 

1-950 
12.685 
7.800 

I2.OIO 
1.5222 
0.8245 
0.6977 
3-9630 

4-3379 
0.2239 

P.  ct. 

1.940 
11.858 
7.504 

I2.OOO 
1.4230 
0.7346 
0.6884 
3.9100 
4-2454 
0.1552 

Total  gluten  in  the  flour.         .       

Part  of  gluten  insoluble  in  alcoh< 
Per  cent  of  nitrogen  in  gluten  .  . 

51  

Total  nitrogen  in  gluten  in  per  ct 
Total  nitrogen  in  residue  of  glut 
Total  nitrogen  extracted  by  alcol 
Gliadin  (N  X  5.68),  assuming  17.1 
Gliadin  by  direct  weighing.  .  .  . 

int  of  flour  

en  insoluble  in 
10! 

alcohol  

3o  per  cent  nitrogen  in  gliadin.  . 

Nitrogen  in  sediment  from  washi 

ng  gluten  

Spring  wheat. 

Winter  wheat. 

Nitrogen. 

Protein. 

Nitrogen. 

Protein. 

Glutenin  

P.  ct. 
0.8245  X  5-68 
0.6077  X  5-68 
0.1248 
0.0657 
0.0453 
0.0341 
0.2239  X  5-68 

P.ct. 

4-683 

3-963 
0.624 
0.391 
0.269 
0.213 
1.272 

P.  ct. 

0.7346  X  5-68 
0.6884  X  5-68 
0.1148 
0.0603 
0.0379 
0.0791 
Q.I552X  5-68 

P.  ct. 
4-173 
3.910 
0.625 

o-359 
0.223 

0.432 
0.881 

Gliadin  

Globulin  

Albumin  

Coagulum  

Proteose  

From  H2O  washings  of  gluten  .  . 

2.0050 

II-4I5 

1.8703 

10  603 

THE    FORMATION    OF    GLUTEN. 

Wheat,  so  far  as  known,  is  the  only  plant  whose  seeds  contain  protein 
matter  separable  in  a  coherent  form  from  the  other  constituents  by  washing 
with  water.  When  ground  fine  and  mixed  with  a  suitable  quantity  of  water, 
it  yields  a  dough  from  which  a  light,  porous  bread  can  be  made.  The  im- 
portance of  this  fact  in  bread-making  is  so  great  that  considerable  attention 
has  been  paid  to  gluten  by  the  chemists  who  have  studied  wheat  proteins. 

Reference  has  already  been  made  (p.  45)  to  the  statements  of  Weyl  & 
Bischoff  that  the  protein  matter  of  the  wheat  kernel  is  chiefly  a  globulin  sub- 
stance, which  in  contact  with  water  undergoes  a  change,  presumably  through 


104  THE   PROTEINS   OF  THE    WHEAT    KERNEL. 

the  influence  of  a  ferment  by  which  gluten  results.  If  the  statements  of 
these  investigators  are  examined  no  evidence  will  be  found  to  support  their 
view.  What  their  reasons  were  for  concluding  that  ' '  myosin ' '  formed 
nearly  all  the  protein  of  the  wheat  kernel  does  not  appear.  In  view  of 
the  results  obtained  by  the  writer,  this  statement  is  certainly  erroneous. 
Direct  treatment  of  the  meal  with  alcohol  yielded  extracts  containing  gliadin 
in  exactly  the  same  amount  as  obtained  from  the  gluten  made  from  an  equal 
quantity  of  flour,  and  extraction  of  either  flour  or  gluten  with  alcohol,  after 
complete  exhaustion  with  sodium-chloride  solution,  also  gave  the  same  pro- 
portion of  gliadin.  This  substance  must  therefore  have  existed  in  the  seed, 
and,  as  it  forms  one-half  of  the  gluten,  it  leaves  the  other  half  only  as 
possibly  derived  from  a  globulin  body  through  the  influence  of  a  ferment. 
If  Weyl  &  Bischoff's  view  were  correct,  treatment  of  the  flour  with  10  per 
cent  salt  solution  ought  to  alter  the  character  and  quantity  of  the  gluten 
obtained,  if  not  altogether  to  prevent  its  formation.  This  is  not  so,  for  the 
usual  amount  of  gluten  can  readily  be  obtained  from  flour  made  into  dough 
with  loper  cent  sodium-chloride  solution  and  then  washed  with  the  same 
until  starch  is  removed. 

Weyl  &  Bischoff  next  state  that  "  with  the  aid  of  a  15  per  cent  rock-salt 
solution  the  flour  was  extracted  until  no  protein  could  be  detected  in  the 
extract;  the  residue  of  the  meal  kneaded  with  water  then  gave  no  gluten. 
If  the  globulin  substance  is  extracted,  no  formation  of  gluten  takes  place. ' '  It 
has  been  found  that  this  is  true  if  the  flour  is  stirred  up  with  a  large  quantity 
of  salt  solution,  extracted  repeatedly  with  fresh  quantities  of  the  same  solu- 
tion until  no  more  protein  is  dissolved,  and  the  excess  of  solution  removed 
by  allowing  the  residue  to  drain  on  a  filter  as  completely  as  possible.  If, 
however,  wheat  flour  is  mixed  at  first  with  just  sufficient  salt  solution  to 
make  a  firm  dough,  this  dough  may  be  washed  indefinitely  with  salt  solution, 
and  will  yield  gluten  as  well  and  as  much  as  if  washed  with  water  alone. 
This  difference  is  due  to  the  fact  that  when  large  quantities  of  salt  solution 
are  applied  at  once  the  flour  fails  to  unite  to  a  coherent  mass  and  can  not  after- 
ward be  brought  together,  as  is  possible  when  treated  with  smaller  quantities 
of  solution. 

Weyl  &  Bischoff  then  compare  the  formation  of  gluten  to  that  of  blood- 
fibrin  from  fibrinogen  under  the  influence  of  a  ferment. 

Sidney  Martin  next  advanced  a  somewhat  similar  theory  of  the  formation 
of  gluten  from  the  proteins  contained  in  the  seed.  He  states  that  alcohol 
extracts  from  gluten  but  one  protein  substance  ;  that  this  is  soluble  in  hot 
water,  but  not  in  cold,  and  he  therefore  calls  it  an  insoluble  phytalbumose. 

The  residue  of  the  gluten  not  dissolved  by  alcohol  is  uncoagulated  pro- 
tein, if  the  alcohol  has  not  been  allowed  to  act  too  long.  This  substance  he 


EXPERIMENTAL.  105 

names  gluten-fibrin.  Martin  further  says  that  gluten  dissolves  almost  com- 
pletely in  0.2  per  cent  hydrochloric  acid  or  0.2  per  cent  potassium-hydroxide 
solution,  leaving  a  small  residue  of  fat.  The  solution  gives  a  copious  pre- 
cipitate when  neutralized,  but  the  supernatant  liquid  still  contains  a  quantity 
of  protein  which  is  the  dissolved  insoluble  albumose.  The  whole  of  the 
gluten-fibrin  is  reprecipitated  by  neutralization — that  is,  it  is  wholly  con- 
verted into  an  albuminate. 

Martin  states  that  by  extracting  flour  with  76  to  80  per  cent  alcohol  only 
fat  is  removed.  This  statement  is  certainly  erroneous,  for  the  writer  has 
never  failed  in  many  experiments  thus  to  extract  this  substance  (gliadin) 
from  the  flour,  and  that,  too,  in  the  same  amount  and  of  the  same  properties 
and  composition  as  from  the  gluten. 

Martin  concludes  that  insoluble  albumose  is  not  present  as  such  in  the 
flour.  He  then  says : 

Before  proceeding  to  mention  its  precursor,  it  will  be  well  to  state  that  10  per  cent 
sodium-chloride  solution  extracts  from  flour  a  large  quantity  of  globulin  and  of  albumose. 
This  globulin  is  of  the  myosin  type,  coagulating  between  55°  and  60°  C. ,  and  precipitated 
by  saturation  with  sodium  chloride  and  ammonium  sulphate.  Both  the  globulin  and 
albumose  are  present  in  a  much  smaller  quantity  in  the  watery  extract  of  the  flour. 

From  this  it  is  evident  that  Martin  has  fallen  into  the  same  error  as  Weyl 
&  Bischoff,  mistaking  the  albumin  for  a  myosin-like  globulin,  and  being 
greatly  misled  as  to  its  amount.  Continuing,  Martin  says  : 

The  direction  of  the  evidence  is  to  show  that  the  insoluble  albumose  is  formed  from 
the  soluble.  Moreover,  I  think  that  the  globulin  is  transformed  into  the  gluten-fibrin, 
for  I  have  been  able  to  obtain  from  the  globulin  in  solution  a  body  having  the  same 
reactions  as  the  gluten-fibrin. 

What  this  evidence  is  which  by  its  direction  shows  that  the  insoluble 
albumose  is  derived  from  the  soluble  is  not  clear,  and  Martin  makes  no 
further  statements  on  this  point.  That  a  body  should  be  obtained  from  the 
solution  containing  the  globulin  which  had  the  same  reactions  as  the 
"  gluten- fibrin  "  is  not  surprising,  for  the  insoluble  products  derived  from 
nearly  all  globulins  have  no  characteristic  reactions,  being  merely  soluble 
in  dilute  acids  and  alkalis  and  precipitated  by  neutralization  in  the  same 
way  as  "  gluten-fibrin."  Martin  then  states  his  theory  of  the  formation  of 
gluten  in  the  following  scheme  : 

PI   .      /Gluten-fibrin — precursor,  globulin. 

~  1  Insoluble  albumose — precursor,  soluble  albumose. 

This  can  not  be  a  correct  representation  of  the  formation  of  gluten,  for  it 
has  been  shown  to  be  founded  on  two  erroneous  observations — first,  that 
alcohol  does  not  extract  protein  matter  from  the  flour  when  applied  directly, 
and,  second,  that  at  least  one-half  the  protein  matter  of  the  seed  is  a  myosin- 
like  globulin. 


106  THE  PROTEINS  OF  THE;  WHEAT  KERNEL. 

The  results  obtained  by  the  author  and  described  in  this  paper  have  led 
to  the  conclusion  that  no  ferment-action  is  involved  in  the  formation  of 
gluten  ;  that  but  two  protein  substances  are  contained  in  the  gluten,  glu- 
tenin  and  gliadin,  and  that  these  exist  in  the  wheat  kernel  in  the  same 
form  as  in  the  gluten,  except  that  in  the  latter  they  are  combined  with 
water  in  an  amount  equal  to  about  twice  the  weight  of  the  dried  protein. 
The  reasons  for  this  opinion  are,  first,  that  alcohol  extracts  the  same  protein 
and  in  the  same  amount,  whether  applied  directly  to  the  flour,  to  the  gluten, 
or  to  flour  previously  extracted  with  10  per  cent  sodium-chloride  solution  ; 
second,  that  0.2  per  cent  potassium-hydroxide  solution  extracts  glutenin  of 
uniform  composition  and  properties  from  flour  which  has  been  extracted  with 
alcohol  or  with  10  per  cent  sodium -chloride  brine  and  then  with  alcohol  as 
it  extracts  from  gluten  which  has  been  exhausted  with  alcohol. 

Both  glutenin  and  gliadin  are  necessary  for  the  formation  of  gluten, 
as  may  be  seen  from  the  following  experiments  :  A  portion  of  flour  was 
washed  completely  free  from  gliadin  by  means  of  alcohol  of  o.  90  sp.  gr. , 
next  with  stronger  alcohol,  finally  with  absolute  alcohol,  and  air-dried. 
The  residue  was  then  rubbed  up  fine  until  all  lumps  were  removed,  and 
water  carefully  added  and  a  dough  made  of  the  mass.  A  tolerably  coherent 
dough  was  thus  obtained,  but  much  less  elastic  and  tough  than  that  pro- 
duced from  the  untreated  flour.  This  dough  was  then  washed  with  water 
on  a  sieve,  using  every  precaution  to  obtain  a  gluten,  but  none  was  formed. 

In  another  experiment  7.5  grams  of  very  finely  ground  air-dried  gliadin 
were  mixed  intimately  with  70  grams  of  fine  corn-starch  and  distilled  water 
added.  A  plastic  dough  was  thus  produced,  but  it  had  no  toughness.  On 
adding  a  little  10  per  cent  sodium-chloride  solution,  the  dough  became  at 
once  tough  and  elastic.  This  was  then  washed  with  great  care  on  a  sieve 
with  cold  water,  a  little  10  per  cent  sodium-chloride  solution  being  added 
from  time  to  time,  but  in  spite  of  every  precaution  no  gluten  was  obtained. 

The  following  experiment  shows  that  the  gliadin  used  was  capable  of 
forming  gluten  when  glutenin  was  present,  and  also  that  salts  have  a  marked 
influence  on  the  toughness  of  the  resulting  dough.  Two  portions  of  flour 
weighing  100  grams  each  were  taken,  and  after  adding  5  grams  of  gliadin  to 
one  both  were  made  into  dough  with  the  same  quantity  of  water.  The 
two  doughs  presented  marked  differences.  That  to  which  gliadin  had  been 
added  was  much  tougher  and  more  yellow  than  the  other.  They  were  then 
washed  with  water  as  long  as  starch  separated.  The  gluten  was  dried 
superficially  by  wiping  with  a  cloth  and  weighed  in  the  moist  state.  That 
from  i oo  grams  of  flour  to  which  5  grams  of  gliadin  had  been  added  weighed 
44-55  grams ;  that  from  100  grams  of  flour  alone  weighed  27.65  grams. 
The  moist  glutens  were  dried  at  1 10°  to  constant  weight,  and  both  yielded 


EXPERIMENTAL.  107 

the  same  proportion  of  dry  gluten,  viz,  34.6  per  cent.  The  yield  of  dry 
gluten  was  accordingly  in  the  first  case  15.41  grams  and  in  the  second  9.56 
grams.  The  difference,  5.85  grams,  shows  that  the  added  gliadin  was  fully 
recovered  in  the  gluten. 

The  figures  show  that  these  proteins  combine  with  about  twice  their 
weight  of  water  in  forming  gluten.  The  fact  that  the  added  gliadin  entered 
so  readily  and  completely  into  the  formation  of  gluten  indicates  that  it 
exists  in  the  seed  as  such  and  undergoes  no  chemical  change  during  extrac- 
tion and  drying. 

The  properties  observed  in  testing  the  separated  gliadin  show  how  it  acts 
in  forming  gluten  and  explain  many  of  the  points  observed  by  others  and 
attributed  to  a  ferment-action. 

When  treated  with  distilled  water  in  small  amount,  the  fine-ground  air-dry 
gliadin  at  once  forms  a  sticky  mass,  which,  on  adding  more  distilled  water, 
dissolves  to  a  turbid  solution.  If,  however,  a  very  little  sodium  chloride  is 
added  to  distilled  water  and  this  applied  to  gliadin  that  has  been  first 
moistened  with  pure  water,  a  very  coherent,  viscid  mass  results,  which 
adheres  to  everything  it  touches  and  can  be  drawn  out  into  long  threads. 
If  the  gliadin  is  moistened  with  10  per  cent  sodium-chloride  solution  and  then 
treated  with  a  larger  quantity  of  this  solution,  the  substance  unites  to  a 
plastic  mass,  which  can  be  drawn  out  into  sheets  and  strings,  but  is  not 
adhesive.  From  this  it  is  evident  why  Ritthausen,  in  washing  flours  which 
gave  a  fluid  gluten,  obtainable  only  in  small  quantity  and  with  great  diffi- 
culty, found  that  the  addition  of  calcium  sulphate  to  the  wash-water  ren- 
dered the  gluten  much  more  coherent  and  easily  obtainable.  The  gliadin  is 
thus  proved  to  be  the  binding  material  which  causes  the  particles  of  flour  to 
adhere  to  one  another,  thus  forming  a  dough.  But  the  gliadin  alone  is  not 
sufficient  to  form  gluten,  for  it  yields  a  soft  and  fluid  mass,  which  breaks  up 
entirely  on  washing  with  water.  The  insoluble  glutenin  is  probably  essen- 
tial by  affording  a  nucleus  to  which  the  gliadin  adheres  and  from  which  it 
is  not  mechanically  carried  away  by  the  wash-water. 

The  behavior  of  the  gliadin  toward  10  per  cent  sodium-chloride  solution 
shows  why  no  gluten  was  obtained  by  Weyl  &  Bischoff  from  flour  extracted 
with  this  solvent.  The  gliadin  had  under  these  conditions  no  adhesive  quali- 
ties, and  therefore  was  unable  to  bind  the  flour  into  a  coherent  mass.  If, 
however,  the  salt  solution  is  added  in  small  quantities  and  the  flour  kneaded 
and  pressed,  the  particles  are  brought  together  and  then  adhere  tenaciously. 


I08  THE   PROTEINS   OF  THE   WHEAT    KERNEL. 


SUMMARY. 

The  proteins  of  the  wheat  kernel  are  gliadin,  insoluble  in  neutral  aqueous 
solutions,  but  distinguished  from  all  the  others  by  its  ready  solubility  in 
neutral  70  per  cent  alcohol ;  glutenin,1  a  protein  having  a  similar  elementary 
percentage  composition  to  gliadin,  soluble  in  very  dilute  acid  and  alkaline 
solutions,  but  insoluble  in  dilute  alcohol  or  neutral  aqueous  solutions  and 
yielding  a  wholly  different  proportion  of  decomposition  products  when  boiled 
with  strong  acids ;  leucosin,  an  albumin-like  protein,  freely  soluble  in  pure 
water  and  coagulated  by  heating  its  solution  to  50°  to  60° ;  a  globulin  similar 
in  composition  and  properties  to  many  globulins  found  in  other  seeds,  and 
one  or  more  proteoses  which  are  present  in  very  small  quantity.  It  has 
also  been  shown  that  the  proteins  obtained  from  the  embryo  of  the  wheat 
are  the  globulin,  albumin,  and  proteose  above  mentioned,  and  that!  these 
form  nearly  all  of  the  protein  substance  of  this  part  of  the  seed.  It  thus 
appears  that  these  three  proteins  are  contained  chiefly  in  the  embryo,  and 
that  gliadin  and  glutenin  form  nearly  the  whole  of  the  proteins  of  the  endo- 
sperm, or  over  80  per  cent  of  the  total  protein  matter  of  the  seed. 

It  is  possible  that  a  part  of  the  albumin,  globulin,  and  perhaps  minute 
quantities  of  the  proteose  are  contained  also  in  the  endosperm,  for  these 
proteins  are  always  found  in  flour  from  which,  in  the  milling  process,  the 
embryo  is  very  nearly  completely  separated.  The  uncertainty,  however, 
as  to  the  completeness  of  this  separation  makes  it  questionable  whether  or 
not  the  small  amount  of  these  proteins  found  in  the  best  flour  is  not  due  to 
the  presence  of  more  or  less  of  the  embryo  that  escaped  separation  in  the 
milling  process. 

The  flour  of  wheat  differs  from  that  of  other  seeds  in  forming  a  dough 
when  moistened  with  sufficient  water,  which,  when  washed  with  more  water, 
loses  its  starch,  and  finally  yields  a  tough  elastic  mass,  long  known  as  wheat 
gluten.  This  gluten  contains  the  greater  part  of  the  protein  matter  of  the 
seed,  together  with  a  little  starch,  fat,  lecithin,  and  phytocholesterin,  and 
possibly  some  carbohydrate  substance  or  substances  of  as  yet  unknown  char- 
acter. These  non-protein  substances  are  probably  not  united  with  one  another 
in  the  gluten,  but  are  mechanically  mixed.  The  quantity  of  starch  that 
remains  in  the  gluten  depends  on  the  thoroughness  of  the  washing,  while 
the  other  substances  owe  their  presence  largely  to  their  insolubility  in  water. 
The  chief  constituents  of  the  gluten  are  the  two  proteins,  gliadin  and  glu- 
tenin, the  relative  proportions  of  which  vary  with  the  variety  of  wheat  from 

1  This  is  the  protein  which  Ritthausen  called  "gluten-casein." 


SUMMARY.  IO9 

which  the  flour  is  made.  The  character  of  the  gluten  and  the  commercial 
value  of  the  flour  depend  to  a  large  extent  on  the  proportion  of  gliadin  to 
glutenin.  <- 

In  the  moist  gluten  these  proteins  are  present  combined  with  about  twice 
their  weight  of  water,  which  is  gradually  lost  on  exposure  to  dry  air  or  at  an 
elevated  temperature. 

The  gliadin  and  glutenin  are  present  as  such  in  the  seed  and  are  not,  as 
was  formerly  supposed,  derived  from  other  protein  substances  through  the 
action  of  an  enzyme.  This  is  shown  by  the  fact  that  they  may  be  obtained 
directly  from  the  flour  by  the  same  treatment  as  that  which  yields  them  from 
the  gluten  and  under  conditions  which  preclude  the  action  of  an  enzyme. 

GUADIN. 

Gliadin  is  the  most  important  of  the  five  proteins  above  mentioned,  not 
only  on  account  of  its  influence  on  the  character  of  the  gluten,  and  therefore 
on  the  quality  of  the  flour  for  domestic  purposes,  but  also  on  account  of  its 
unusual  physical  properties  and  chemical  constitution. 

Gliadin,  unlike  most  other  protein  substances,  is  freely  soluble  in  relatively 
strong  ethyl  alcohol.  Although  gliadin  is  wholly  insoluble  in  absolute  alcohol 
and  but  slightly  soluble  in  water,  it  dissolves  in  dilute  alcohol,  the  solubility 
increasing  with  increasing  concentration  of  alcohol  until  a  certain  strength 
is  reached,  when  the  solubility  diminishes  until,  by  absolute  alcohol,  it  is  no 
longer  dissolved.  Exactly  what  strength  of  alcohol  dissolves  the  largest 
proportion  of  gliadin  has  never  been  determined,  but  the  maximum  solubility 
is  attained  with  about  70  per  cent  of  alcohol  by  volume. 

Gliadin  is  also  soluble  in  other  alcohols,  as  methyl,  propyl,  and  benzyl 
alcohols,  in  phenol  and  paracresol,  and  also  in  glacial  acetic  acid. 

Gliadin  is  somewhat  soluble  in  pure  water,  but  less  so  in  water  containing 
salts,  though  not  wholly  insoluble  in  solutions  containing  10  per  cent  of 
sodium  chloride.  Very  dilute  acid  or  alkaline  solutions  dissolve  gliadin  to 
solutions  which,  by  neutralizing,  yield  the  gliadiu  apparently  unchanged.  By 
stronger  solutions  of  acids  or  alkalis  the  gliadin  is  altered,  as  is  the  case 
with  all  other  native  proteins. 

All  the  usual  color  tests  given  by  other  protein  substances  are  obtained 
with  gliadin,  which  therefore  contains  the  groups  that  give  rise  to  these  re- 
actions. Gliadin  is  much  less  easily  converted  into  insoluble  products  than 
are  most  other  proteins.  Its  solution  in  70  per  cent  alcohol  can  be  boiled 
for  an  indefinite  time,  and  even  concentrated  until  much  of  the  alcohol  has 
been  removed,  without  forming  insoluble  products.  On  heating  with  very 
weak  alcohol  or  with  water  this  protein  is  gradually  altered  and  becomes 
insoluble  in  stronger  alcohol,  but  the  coagulated  gliadin  thus  formed  is  in 


no 


THE;  PROTEINS  OF  THE;  WHEAT  KERNEL. 


appearance  unlike  the  heat  coagulum  formed  by  most  other  proteins.  Pro- 
teins of  similar  behavior  to  gliadin  are  found  in  the  seeds  of  other  cereals, 
such  as  rye,  barley,  maize,  oats,  and  sorghum.  That  found  in  rye  is  prob- 
ably identical  with  the  gliadin  of  wheat,  for  a  rigid  comparison  has  not  yet 
revealed  any  difference.  Both  have  the  same  ultimate  composition,  the 
same  solubility,  the  same  physical  properties,  and  yield  the  same  amount  of 
ammonia  and  glutaminic  acid  on  hydrolysis.  The  identity  of  the  two,  how- 
ever, is  not  certain,  and  with  our  present  knowledge  can  not  be  established. 
The  proteins  of  the  other  cereals  above  mentioned  are  distinctly  different, 
though  similar,  proteins. 

Proteins  characterized  by  such  ready  solubility  in  strong  alcoholic  solu- 
tions as  are  those  found  in  these  cereals  have  not  been  obtained  from  the 
seeds  of  any  other  plants. 

The  ultimate  composition  of  gliadin  has  been  fixed  within  narrow  limits 
by  the  accordant  analyses  of  several  investigators  as  follows  : 


Giinsberg. 

Ritthausen. 

Osborne  & 
Voorhees. 

Nasmith. 

Konig  & 
Rintelen. 

Carbon  

P.ct. 

52.67 

P.ct. 
52.76 

P.ct. 
52.72 

P.ct. 

52.  1Q 

P.ct. 

52.70 

Hydrogen  .  . 
Nitrogen  
Sulphur  

6.83 
17.62 

7.10 
18.01 
o.8s 

6.86 
17.66 

I.Ot 

6.84 

17-47 
1.  12 

7.62 

17.77 

O  Q5 

Oxygen. 

22.88 

21.28 

21.  71 

22.18 

20.96 

IOO.OO 

IOO.OO 

IOO.OO 

IOO.OO 

100  00 

Gliadin  yields  on  hydrolysis  a  larger  amount  of  ammonia,  glutaminic  acid, 
and  proline  than  any  other  protein  yet  examined,  no  glycocoll  or  lysine,  and 
a  relatively  small  amount  of  histidine  and  arginine,  as  the  following  analysis 
shows  : 

Products  of  hydrolysis  of  gliadin. 


P.ct. 

Glycocoll o.oo 

Alanine 2.00 

Amino-valerianic  acid 0.21 

Leucine 5.61 

Proline 7.06 

Phenylalanine 2.35 

Aspartic  acid 0.58 

Glutaminic  acid 37-33 

Serine o.  13 


P.ct. 

Tyrosine 1.20 

Cystine 0.45 

Lysine o.oo 

Histidine 0.61 

Arginine 3. 16 

Ammonia 5.11 

Tryptophane present 

Total 65.81 


Although  gliadin  gives  a  strong  Molisch  reaction,  which  is  commonly  con- 
sidered to  indicate  the  presence  of  a  carbohydrate  group,  it  gives  no  f urfurol 
when  distilled  with  hydrochloric  acid,  as  does  ovalbumin,  which  has  been 


SUMMARY. 


Ill 


proved  to  contain  such  a  group.  Other  evidence  than  that  given  by  the 
Molisch  reaction  is  required  before  the  presence  of  a  carbohydrate  group  in 
this  protein  can  be  assumed. 

Nearly  two-thirds  of  the  total  sulphur  in  gliadin  is  split  off  as  sulphide 
by  boiling  with  alkalis,  which  would  indicate  the  possibility  that  all  the 
sulphur  is  contained  in  a  cystine  complex.  The  amount  of  cystine  isolated 
is  far  below  that  required  by  such  an  assumption.  The  determination, 
however,  of  this  substance  is  not  quantitative,  and  the  figure  given  does  not 
necessarily  prove  that  all  of  the  sulphur  may  not  be  contained  in  a  cystine- 
yielding  complex. 

The  specific  rotation  of  gliadin  in  80  per  cent  by  volume  ethyl  alcohol,  ac- 
cording to  determinations  by  the  writer,  which  agree  closely  with  those  of 

20° 
others,  is  («)  —  =  —92.3°. 

The  specific  rotation  in  solvents  of  alcoholic  nature  has  been  recently 
determined  by  Mathewson,  who  gives  the  following  data  : 


Methyl  alcohol,  70  per  cent.  —  95.65° 
Ethyl  alcohol,  70  per  cent.  —  9i-95c 
Ethyl  alcohol,  60  per  cent.  —  96.66° 
Ethyl  alcohol,  50  per  cent.  —  98.45° 
Propyl  alcohol,  60  per  cent.  — ioi.ioc 


Phenol,  70  per  cent —  123.15° 

Phenol,  anhydrous — 131.77° 

Paracresol — 121.00° 

Benzyl  alcohol —   53. 10° 

Glacial  acetic  acid —   78.60° 


The  amount  of  gliadin  in  different  varieties  of  wheat  differs  to  a  large 
extent,  as  shown  by  a  considerable  number  of  determinations  that  have  been 
recorded.  Thus  Teller  found  in  a  sample  of  Canadian  white  winter  wheat 
2.74  per  cent,  in  one  of  Oregon  white  winter  wheat  2.85  per  cent,  and 
in  a  red  spring  wheat  from  South  Dakota  8.15  per  cent.  In  other  varieties 
of  wheat  he  and  others  have  found  quantities  falling  between  these  figures. 

Although  the  total  quantity  of  gliadin  in  the  sample  of  red  wheat  above 
mentioned  was  nearly  three  times  as  great  as  in  that  of  the  white  wheat,  its 
percentage  of  the  total  proteins  was  much  more  uniform,  being  42.5  and  34 
per  cent  respectively.  This  appears  to  be  generally  true  for  most  of  the 
wheats  that  have  been  examined. 

Thus  Shepard  has  found  in  13  samples  of  durum  wheat  that  the  gliadin 
formed  from  40.2  to  48.6  per  cent  of  the  total  protein  (N  X  5.7),  in  9  sam- 
ples of  Northwestern  spring  wheat  from  44.2  to  49  per  cent,  in  4  samples 
of  Kansas  hard  winter  wheat  from  40  to  50.7  per  cent,  and  in  3  samples  of 
soft  winter  wheat  from  40  to  47.4  per  cent.  The  figures  given  by  others  are 
similar,  though  a  few  fall  between  somewhat  wider  limits.  It  would  seem 
safe  to  say  that  in  the  majority  of  wheats  the  gliadin  forms  from  40  to  50 
per  cent  of  the  total  proteins,  and  that  its  absolute  amount  depends  chiefly 
on  the  total  protein  content  of  the  seed. 


112  THE   PROTEINS   OF  THE   WHEAT    KERNEL. 

GLUTENIN. 

This  protein  was  first  described  by  Taddei  under  the  name  of  zymom. 
I,iebig,  as  well  as  Dumas  &  Cahours,  named  it  "  plant-fibrin  ; "  Ritthausen 
called  it  ' '  gluten-casein  ; ' '  Weyl  &  Bischoff  considered  it  to  be  an  albumi- 
nate  form  of  a  myosin-like  globulin.  On  the  ground  of  priority  and  the  fact 
that  the  relations  to  animal  proteins,  which  gave  rise  to  the  various  names 
subsequently  applied  to  this  body,  have  been  proved  to  have  no  foundation, 
it  would  be  desirable  to  return  to  Taddei 's  original  name  and  in  future  call 
this  protein  zymom.  Unfortunately  this  name  is  derived  from  the  Greek 
word  Ct>/«7,  a  ferment,  and  as  the  results  of  this  and  all  other  subsequent 
investigations  of  this  subject  show  that  the  supposed  ferment-changes  do  not 
occur  in  the  formation  of  gluten,  it  seems  appropriate  to  call  it  glutenin,  a 
name  suggested  by  S.  W.  Johnson.  Glutenin  is  next  in  importance  to 
gliadin,  for  in  most  varieties  of  flour  it  is  present  in  nearly  equal  amount.  It 
is  so  nearly  insoluble  in  water  and  alcohol  that  it  is  a  question  whether  the 
slight  traces  that  are  dissolved  by  these  solvents  from  carefully  purified  prep- 
arations are  not  due  to  traces  of  gliadin,  which  it  is  very  difficult  to  separate 
from  it.  In  hot  dilute  alcohol  glutenin  is  slightly  soluble  and  separates 
from  such  solutions  on  cooling.  When  freshly  precipitated  and  in  the 
hydrated  condition,  it  is  very  readily  dissolved  by  extremely  dilute  acids  or 
alkalis  and  is  precipitated  from  such  solutions  on  neutralization.  Glutenin 
can  be  separated  from  the  other  constituents  of  the  seed  only  by  solution  in 
dilute  alkalis  or  acids,  and  it  is  necessary  to  filter  the  solutions,  from  which 
it  is  finally  precipitated,  perfectly  clear  in  order  to  separate  the  associated 
non- protein  substances.  This  filtration  is  accomplished  with  great  difficulty 
unless  it  is  preceded  by  thorough  extraction  of  the  crude  glutenin  with 
alcohol  and  ether,  whereby  fats  and  lecithins  are  removed. 

Glutenin  contains  all  the  groups  which  give  rise  to  the  usual  color  reac- 
tions of  the  proteins.  No  protein  similar  to  glutenin  in  physical  and  chem- 
ical properties  has  yet  been  found  in  any  other  seed.  Although  its  ultimate 
composition  is  nearly  the  same  as  gliadin,  the  proportion  of  the  various 
products  of  hydrolysis  which  it  yields  is  very  different,  as  may  be  seen  from 
the  following  analyses  : 

The  ultimate  composition  of  glutenin. 

P.ct. 

Carbon 52.34 

Hydrogen 6.83 

Nitrogen 17-49 

Sulphur i. 08 

Oxygen 22.26 


SUMMARY. 


The  following  products  of   hydrolysis   have  been  obtained  by  boiling 
glutenin  with  strong  hydrochloric  acid  : 

Products  of  hydrolysis  of  glutenin. 


P.ct. 

Glycocoll 0.89 

Alanine 4.65 

Arnino-valerianic  acid 0.24 

Leucine 5.95 

a-proline 4.23 

Phenylalanine 1.97 

Aspartic  acid 0.91 

Glutaminic  acid 23.42 

Serine 0.74 


P.ct. 

Tyrosine ...     4-25 

Cystine 0.02 

L,ysine 1.92 

Histidine 1.76 

Arginine 4-72 

Ammonia 4-01 

Tryptophane present 

59.66 


Glutenin  is  qualitatively  distinguished  from  gliadin  by  yielding  both 
glycocoll  and  lysine,  and  quantitatively  by  yielding  less  proline,  glutaminic 
acid,  and  ammonia  and  more  alanine,  tyrosine,  and  arginine.  In  other 
respects  the  differences  are  not  great. 

The  amount  of  glutenin  varies  greatly  in  different  samples  of  wheats,  but 
usually  forms  about  40  per  cent  of  the  total  protein  of  the  seed.  Teller  has 
found  in  a  sampleof  Canadian  white  winter  wheat  3. 64  per  cent  of  glutenin  and 
in  an  Oregon  white  winter  wheat  3.82  per  cent,  while  in  a  red  spring  wheat 
from  South  Dakota  he  found  8.04  per  cent.  Although  the  total  amount  of 
glutenin  in  these  wheats  was  very  different,  the  relative  amounts  were  nearly 
the  same,  being  45.2  and  46.5  percent  of  the  total  proteins  in  the  white 
winter  wheats  and  42  per  cent  in  the  red  spring  wheat.  Other  winter 
wheats  differed  but  little  from  other  spring  wheats  both  in  the  total  and 
relative  amount  of  glutenin  which  they  contained.  In  most  of  the  wheats 
that  he  examined  the  glutein  formed  from  40  to  45  per  cent  of  the  total 
proteins.  Shepard,  who  analyzed  a  much  larger  number  of  samples,  found 
a  wider  variation  in  the  relative  proportion  of  glutenin,  from  34.2  to  46.8 
per  cent  of  the  total  proteins,  and  a  narrower  range  in  its  total  amount,  the 
latter  falling  between  3.41  and  7.10  per  cent. 

LEUCOSIN. 

L,eucosin  forms  about  0.3  to  0.4  per  cent  of  the  wheat  kernel.  Although 
the  entire  seed  contains  but  little  leucosin,  the  embryo  contains  a  relatively 
large  proportion,  since  about  10  per  cent  of  the  commercial  "  germ  meal  " 
consists  of  this  protein.  As  this  "  germ  meal  "  contains  more  or  less  of  the 
endosperm  and  outer  coats  of  the  seed,  the  embryo  contains  somewhat  more 
than  10  per  cent.  L,eucosin  is  an  albumin,  for  it  is  soluble  in  pure  water 
and  coagulated  by  heating  its  solution.  It  has  been  mistaken  by  several 
observers  for  a  myosin-like  globulin,  owing  to  the  fact  that  its  temperature 
of  coagulation  falls  near  to  that  of  such  globulins  found  in  animal  tissues, 


114  THE   PROTEINS   OF   THE   WHEAT    KERNEL. 

and  also  to  the  fact  that  its  solutions  are  precipitated  by  saturating  them  with 
sodium  chloride  or  with  magnesium  sulphate.  That  it  is  soluble  in  pure 
water  and  not  held  in  solution  by  small  quantities  of  salts  is  shown  by  the 
fact  that  a  solution  containing  a  considerable  quantity  of  leucosiu  when  sub- 
jected to  prolonged  dialysis  and  then  evaporated  to  dryness,  and  the  leucosin 
burned  off  at  a  low  temperature  left  a  residue  of  mineral  matters  weighing 
less  than  a  milligram.  Leucosin  is  much  more  readily  precipitated  by  am- 
monium sulphate  than  are  the  albumins  of  animal  origin,  for  by  adding  an 
equal  volume  of  a  saturated  solution  of  this  salt  to  solutions  containing  leu- 
cosin the  latter  is  almost  completely  precipitated. 

Leucosin  resembles  the  animal  proteins  in  ultimate  composition,  in  the 
proportion  of  its  products  of  hydrolytic  decomposition,  and  in  its  physical 
properties  more  closely  than  it  resembles  most  of  the  seed  proteins  yet  studied. 
It  seems  not  improbable  that  this  is  because  it  is  a  constituent  of  the  tissues 
of  the  embryo,  for  its  physiological  functions  are  unquestionably  different 
from  those  of  the  stored-up  food  proteins  of  the  endosperm. 

Proteins  having  the  same  ultimate  composition  and,  so  far  as  known,  the 
same  properties  as  leucosin  are  found  in  the  seeds  of  other  cereals.  Whether 
these  are  identical  or  not  can  not  be  determined  by  any  means  now  available. 

The  ultimate  composition  of  leucosin  is  shown  by  the  following  figures, 
which  are  the  average  of  accordant  analyses  of  several  preparations  : 

Composition  of  leucosin. 

P.ct. 

Carbon 53-Q2 

Hydrogen 6.84 

Nitrogen 16.80 

Sulphur 1.28 

Oxygen 22.06 


Leucosin  yields  the  following  amounts  of  the  several  products  of  hydrolysis 
when  boiled  with  strong  hydrochloric  acid  : 

Products  of  hydrolysis  of  leucosin. 

P.  ct.  P.  ct. 


Glycpcoll 0.94 

Alanine 4-45 

Amino-valerianic  acid o.  18 

Leucine n  .34 

a-proline 3 . 18 

Phenylalanine 3.83 

Aspartic  acid 3.35 

Glutatninic  acid 6.73 


Tyrosine 3.34 

Lysine 2 . 75 

Histidine 2.83 

Arginine   : 5.94 

Ammonia 1.41 

Tryptophane present 

50.32 


Leucosin  probably  contains  a  relatively  large  proportion  of  tryptophane, 
for  in  a  series  of  comparative  tests  made  with  a  large  number  of  different 


SUMMARY.  115 

proteins  the  intensity  of  its  glyoxylic  acid  reaction  was  greater  than  that  of 
any  of  the  other  proteins. 

L,eucosin  also  gave  the  strongest  reaction  with  the  Molisch  test  of  any  of  a 
large  number  of  different  proteins  examined  under  like  conditions,  but 
whether  or  not  it  contains  a  carbohydrate  group  must  be  shown  by  other 
evidence. 

THE    GLOBULIN. 

The  seeds  of  wheat  contain  about  o.  6  per  cent  of  a  protein  which  is  insol- 
uble in  water,  but  soluble  in  neutral  saline  solutions.  This  globulin  is  chiefly 
contained  in  the  embryo  from  which  5  per  cent  was  extracted  by  sodium- 
chloride  solution.  The  preparations  of  the  globulin  that  were  made  from 
the  embryo  contained  nucleic  acid,  while  those  from  the  whole  seed  con- 
tained none.  These  nucleic  acid  compounds  from  the  embryo  behaved  like 
nucleates,  for  their  proportion  of  nucleic  acid  was  not  constant,  but  varied 
with  the  conditions  of  preparation.  It  seemed  most  probable  that  the  nucleic 
acid  combines  with  the  basic  protein  to  form  salts  in  the  same  manner  as 
other  acids  are  known  to  do,  and  that  such  combinations  with  the  globulin 
still  retained  the  solubility  characteristic  of  the  globulin.  It  is  quite  possi- 
ble that  other  insoluble  compounds  containing  more  nucleic  acid  may  have 
existed  in  the  embryo  or  have  been  formed  during  extraction,  so  that  the 
amount  of  globulin  obtained  may  not  have  equaled  that  actually  present  in 
the  embryo.  The  fact  that  the  globulin  from  the  whole  seed  was  free  from 
nucleic  acid,  while  that  from  the  embryo  was  not,  was  probably  due  to  the 
presence  of  a  much  larger  proportion  of  protein  insoluble  in  salt  solution  in 
the  whole  seed  compared  with  that  in  the  embryo,  so  that  the  nucleic  acid 
united  with  the  insoluble  protein  instead  of  with  the  globulin,  as  happened 
when  the  embryo  was  extracted. 

This  globulin  is  very  similar  to,  if  not  identical  with,  that  found  in  the 
seeds  of  rye  and  barley.  It  contains  over  18  per  cent  of  nitrogen,  and  re- 
sembles, in  composition  and  properties,  many  of  the  globulins  found  in  large 
proportion  in  many  other  seeds. 

Owing  to  the  difficulty  encountered  in  preparing  large  quantities  of  this 
globulin,  the  products  of  its  hydrolysis  have  not  been  studied. 

Its  ultimate  composition  is  shown  by  the  following  figures,  which  are  the 
average  of  several  analyses  of  preparations  from  the  whole  seed  : 

P.ct. 

Carbon 51-03 

Hydrogen 6.85 

Nitrogen 18.39 

Sulphur 0.69 

Oxygen 23.04 


u6 


THE   PROTEINS   OF  THE    WHEAT    KERNEL. 


This  globulin  is  precipitated  by  saturating  its  solutions  with  magnesium 
sulphate,  but  not  with  sodium  chloride.  Dissolved  in  10  per  cent  sodium- 
chloride  solution,  it  is  partly  coagulated  by  boiling,  but  is  not  coagulated  at 
temperatures  below  100°. 

THE  PROTEOSE. 

The  wheat  kernel  yields  a  very  small  amount  of  proteose  when  extracted 
with  water.  Whether  this  is  an  actual  constituent  of  the  seed  or  is  formed 
from  the  other  proteins  during  its  extraction  and  isolation  was  not  definitely 
ascertained.  This  proteose,  like  leucosin  and  globulin,  is  chiefly  found  in 
the  embryo.  No  preparations  were  made  from  the  entire  seed  in  sufficient 
quantity  to  permit  of  analysis  or  examination  of  its  properties. 

The  proteose  obtained  from  the  embryo  appeared  to  be  a  mixture  of  two 
or  more  substances.  A  part  was  precipitated  by  saturating  its  solution  with 
sodium  chloride ;  a  part  was  not. 

The  analyses  of  these  two  parts  showed  the  low  percentage  of  carbon 
characteristic  of  proteoses  obtained  by  peptic  digestion.  Analyses  of  prepara- 
tions thus  obtained  gave  the  following  results  : 


Proteose  in- 
soluble in 
saturated 
Nad 
solution. 

Proteose 
soluble  in 
saturated 
NaCl 
solution. 

Carbon  

P.ct. 
40.04 

P.ct. 
48.00 

Hydrogen  
Nitrogen  

6.80 
17.08 

6.85 
16  89 

Sulphur  

1.24 

I.IO 

Oxveren.  .  , 

24.Q4 

26.17 

100.00 

100.00 

THE   GLUTEN. 

The  proteins  of  the  wheat  kernel  differ  from  those  that  have  been  found 
in  any  other  seed  by  the  fact  that  they  may  be  largely  separated  as  a  coherent 
elastic  mass  by  washing  the  dough  made  from  the  flour  under  a  gentle  stream 
of  water.  This  protein  mass  has  long  been  known  as  gluten.  It  consists 
chiefly  of  gliadin  and  glutenin  in  combination  with  about  twice  their  weight 
of  water,  together  with  more  or  less  starch,  which  can  not  be  wholly  removed 
by  washing,  and  also  some  fat,  cholesterin,  and  lecithin.  The  non-protein 
constituents  usually  form  about  20  to  25  per  cent  of  the  crude  gluten.  The 
protein  constituents  of  the  gluten,  except  for  the  fact  that  they  are  combined 
with  water,  are  present  in  the  same  form  in  the  gluten  as  in  the  grain. 


SUMMARY.  IIJ 

Their  proportion  is  also  nearly  the  same,  for  practically  the  same  amount  of 
gliadin  can  be  obtained  from  the  gluten  as  from  the  flour,  if  allowance  is 
made  for  the  small  amount  of  gluten  that  is  mechanically  carried  away  in 
the  process  of  washing  out  the  starch.  This  loss  is  largely  made  up  by  the 
presence  of  non-protein  constituents  in  the  gluten,  so  that  the  weight  of  the 
dried  gluten  usually  corresponds  closely  with  the  amount  calculated  for  the 
sum  of  gliadin  and  glutenin,  as  computed  from  the  nitrogen  belonging  to 
these  proteins. 

The  gluten  is  not  formed  from  globulins  by  the  action  of  a  ferment  in  a 
manner  analogous  to  the  formation  of  fibrin  from  fibrinogen,  as  was  asserted 
at  one  time.  The  observations  on  which  this  supposition  was  founded  were 
incorrect. 

The  glutenin  probably  forms  the  nucleus  to  which  the  gliadin  adheres, 
and  thus  binds  the  gluten  proteins  into  a  coherent  elastic  mass. 

Both  gliadin  and  glutenin  are  necessary  for  the  formation  of  gluten,  for  a 
dough  made  with  starch  and  gliadin,  or  one  made  with  flour  from  which  the 
gliadin  has  been  extracted  with  alcohol,  yields  no  gluten  when  washed  with 
water.  That  the  gliadin  is  capable  of  taking  part  in  gluten  formation  under 
such  conditions  is  shown  by  the  fact  that  when  dry  and  finely  ground  glia- 
din was  added  to  wheat  flour  the  amount  of  gluten  obtained  on  subsequently 
washing  out  the  starch  was  increased  by  the  full  amount  of  the  gliadin  that 
was  added. 

THE   NUTRITIVE    VALUE   OF  THE   WHEAT   PROTEINS. 

It  has  recently  been  shown  that  in  the  process  of  digestion  the  protein 
molecule  is  very  largely  broken  down  into  amino-acids,  and  that  the  animal 
forms  from  these,  by  the  processes  of  assimilation,  the  proteins  of  its  blood 
and  tissues.  How  this  change  is  effected  is  not  known,  nor  is  it  known 
whether  the  food  protein  is  converted  into  the  body  protein  and  then  ox- 
idized and  eliminated,  or  is  partly  converted  into  body  protein  and  partly 
burned  directly  in  the  form  of  amino-acids.  It  is  also  a  question  whether 
or  not  the  animal  has  the  power  to  convert  one  amino-acid  into  another,  and 
thus  obtain  material  suitable  for  the  construction  of  its  own  body  protein. 

Directly  connected  with  these  important  problems  are  the  facts  presented 
by  the  determination  of  the  relative  amounts  of  the  different  amino-acids 
yielded  by  the  proteins  of  wheat  flour,  for  these  are  used  in  enormous  quan- 
tities as  food  by  man,  and,  as  an  examination  of  the  analyses  of  their  decom- 
position products  show,  present  marked  differences  from  similar  analyses  of 
all  the  other  food  proteins  thus  far  examined. 

As  gliadin  and  glutenin  together  form  about  85  per  cent  of  the  proteins  of 
wheat  flour,  they  deserve  especial  consideration  in  this  respect.  If  it  is 


THE   PROTEINS   OE  THE   WHEAT    KERNEL. 


assumed  that  these  two  proteins  are  present  in  equal  quantities  in  wheat 
flour,  an  assumption  that  in  most  cases  is  nearly  correct,  the  relative  propor- 
tion of  the  amino-acids  yielded  by  the  proteins  of  wheat  flour  is  closely 
shown  by  the  following  figures,  which  are  the  mean  of  those  found  for 
gliadin  and  glutenin.  In  comparison  with  these  are  given  the  figures,  at 
present  available,  for  some  of  the  other  forms  of  food  protein.  Such  a  com- 
parison is  at  present  unsatisfactory,  owing  to  the  lack  of  data  now  available, 
but  it  will  serve  a  useful  purpose  in  indicating  the  wide  differences  between 
these  food  proteins,  and  will  emphasize  the  importance  of  obtaining  more 
information  in  regard  to  this  question. 

Products  of  hydrolysis  of  some  food  proteins. 


wheat 
gluten. 

Milk 
casein. 

,^gg- 

albumin. 

Zein, 
maize. 

Excel- 
sin, 
Brazil 
nut. 

Phase- 
olin, 
white 
bean. 

Beef 

muscle. 

Halibut 
muscle. 

Glycocoll  

P.  ct. 
r>,/M 

P.ct. 

o  oo 

P.ct. 

O  OO 

P.ct. 

P.ct. 
O.6o 

P.ct. 
O.  SS 

P.ct. 

P.ct. 

Alanine  

-i.-i-i 

o.qo 

2.  IO 

2.^ 

1.  80 

Amino-valerianic  acid. 

O.2^ 

I.OO 

LSI 

I.O4 

L,eucine  .         

S.78 

10.  so 

6.  10 

8.70 

Q.  S6 

Proline  

5.65 

^.10 

2.2*; 

3.65 

2.77 

Phenylalanine  

2.16 

V2O 

4.  4O 

V  SS 

^•2S 

Aspartic  acid 

O.7S 

1.  20 

I.  SO 

3.85 

S.24 

Glutaminic  acid  

30.38 

II.  OO 

Q.  10 

16.87 

12.94 

14-54 

II.  I 

8.9 

Serine  

O.44. 

0.2^ 

0.^8 

Tyrosine                  .  .  . 

2.71 

4.  co 

I.IO 

3.16 

2.17 

Cvstine 

O  24 

0.06 

O.2O 

L/ysine  

O.Q6 

•5.80 

o.oo 

1.64 

3.59 

Histidine            

I.IQ 

2.SQ 

0.81 

1.47 

I.Q7 

Arginine  

7.  Q4. 

4.84 

1.82 

16.02 

4.72 

Ammonia     

•5  "7 
4.  ^6 

I.  QS 

1.63 

3.61 

1.80 

2.06 

Tryptophane 

present 

I.  SO 

present 

o.oo 

present 

present 

Owing  to  the  lack  of  data,  it  is  not  yet  possible  to  compare  the  relative 
proportion  of  the  amino-acids  which  the  food  proteins  yield  except  in  respect 
to  glutaminic  acid,  ammonia,  arginine,  histidine,  and  lysine.  The  amount 
of  glutaminic  acid  which  the  gluten  proteins  yield  is  far  greater  than  that 
yielded  by  any  of  the  other  food  proteins,  with  the  exception  of  gliadin  from 
rye  and  hordein  from  barley.  The  proteins  of  the  legumes  and  nuts  which 
are  used  as  food  yield  from  15  to  20  per  cent  of  glutaminic  acid,  so  that  the 
mean  amount  of  this  ammo-acid  from  the  wheat  protein  is  nearly  twice  as 
large.  The  same  also  is  true  of  ammonia. 

The  proportion  of  arginine  from  wheat  gluten  is  relatively  small  compared 
with  that  from  most  other  seed  proteins,  many  of  which  yield  from  10  to  16 
per  cent  of  this  base. 


SUMMARY.  119 

The  proportion  of  lysine  is  likewise  small,  especially  compared  with  that 
obtained  from  the  leguminous  seeds.  The  amount  of  histidine,  however, 
does  not  differ  very  greatly  from  that  of  the  other  seed  proteins.  What 
significance  these  differences  have  in  respect  to  the  nutritive  value  of  these 
different  proteins  must  be  determined  by  future  investigation,  for  it  has  only 
very  recently  been  discovered  that  such  differences  exist. 

That  a  molecule  of  gliadin  can  have  the  same  nutritive  value  as  one  of 
casein  would  seem  impossible  if  one  molecule  of  food  protein  is  transformed 
into  one  of  tissue  protein,  for  in  the  former  lysine  is  wholly  lacking,  and 
glutaminic  acid,  ammonia,  and  proline  are  in  great  excess  over  the  amount 
required  to  form  any  of  the  tissue  proteins  of  which  we  know.  It  would 
seem  probable  that  either  the  animal  requires  a  variety  of  food,  so  that  the 
relative  proportion  in  which  the  amino-acids  are  available  for  its  use  shall 
correspond  more  nearly  to  its  requirements,  or  that  only  a  small  part  of 
these  amino-acids  are  converted  into  its  tissue  proteins  and  the  rest  oxidized 
as  such.  It  is  possible  that  feeding  experiments  with  proteins  of  known 
character  in  respect  to  the  relative  proportions  of  their  decomposition  prod- 
ucts will  throw  light  on  these  important  questions. 


